Metabolic Arrest Cont

7/27/2019 Metabolic Arrest Cont

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Metabolic Arrest cont.

- Look at post-transitional events after stress

- Some organisms maintain rxns away from eq, which allows them to have control, the so called

irreversible reactions.

- We will look at useful energy in this regard.

Principles of metabolic regulation and control:

- Homeostasis – cells and organisms constitute open, non-eq systems operating at steady-state

- Pathway flux is regulated to meet the needs of the organism

- Reciprocal regulation of opposing pathways

o Glycolysis and gluconeogenesis

o If you don’t reciprocate some pathways there would be a waste of energy = futile cycles.

o They occur in non-glyconeogeniate tissue, G6P is active even though these muscles

don’t take aprt in gluconeogenesis

- Mechanisms of flux control

o Input/outputo Regulation of enzyme activity

o Substrate/Futile cycle

Dam analogy:

- Why organisms control the irreversible rxn and not the ones that are near eq?

- If you don’t control the non-eq rxns, you have a loss of free energy, so you use the dam to

maintain the ubstrate upstream. The near eq reactiosn can go either way given a change in the

substrate, product ratios.

In glycolysis:

- Hexokinase, PFK1 (rate limiting) and Pyruvate Kinase

3 rxns in glycolysis that are far from equilibrium.

Flux through glycolysis are controlled at these points.

In anoxia states, the PK becomes more important.

Large free energy drops, irreversible rxn. Rapid change in flux with small change to enzyme. Rapid

turning on/off is what organisms want to do!

1) PFK-1 glycolysis/gluconeogenesis ctrl point

F6P + ATP –PFK F1, 6 BP – ADP



2) Pyruvate Kinase (PK) – primary control point for transition into anoxic state.

Phophoenolpyruvate (PEP) + ADP – PK Pyruvate + ATP

Regulating Metabolic Pathway Flux

Vmax or Km regulation

- If you were to design a metabolic pathway, such as glycolysis, how would you regulate the rate

of conversion of glucose to pyruvate?

Items to consider:

- Is it necessary to regulate every enzyme in the pathway or just several key enzymes?

- Would you design a system that operated at or near its maximum rate – Vmax under saturating

conditions?

- Would you regulate the pathway at or near the Km values of various substrates for the

corresponding enzymes?

Vmax Regulation – difficult to imagine a system like this.

By def reqs saturating substrate concentrations at all times:

- Results in high resting metabolic rate (max metabolic rate)

- Waste valuable substrate- Need mechanism to add and remove active enzyme to the pathway from an inactive pool

- Only situation where this is known to occur is in muscle where actin-myosin ATPase is kept in an

inactive state by troponin C. binding of Ca allows the release.

Because of these issues –generally organisms don’t choose to use this control.

Km regulation – strategy employed by the vast majority of metabolic pathways including glycolysis

and krebs

If substrate concentrations are near their Km values, then small changes in concentration result in largechanges in reaction rate. Km sites on the steep portion of the curve in MM curves. Very sensitive around

the km to changes in [S]

Importantly, Km regulation allows for high level of allosteric and covalent regulation of reaction rate.

- By altering the binding affinity or sensitivity of an enzyme for its substrate the rxn rate can be

modified.

- Affords a built in autoregulatory mechanism



- Km is the [S] that gives ½ Vmax

Tetramers, cooperativity: allosteric binding will affect the affinity of enzyme for its substrate.

Sigmoidal curves – don’t follow MM kinestics, they have Vmax and Km (called S0.5] affinity V of enzyme

for S, or [S]/2. The effect of activators – decreased Km, increased affinity of S for the enzyme, will take

less substrate to reach Vmax.

PFK 1 Regulators:

ATP = inhibitor (lots of ATP!, don’t need more!)

AMP, ADP = activator (need more ATP!)

Citrate = inhibitor (Krebs is active)

Fructose 2, 6-bisphosphate – MOST POWERFUL ACTIVATOR, PRODUCED BY PFK2!

Allosteric Regulation of PFK 1:

- Low[ATP] = Activator = Reduced Km, but Vmax unchanged!

- High[ATP] = Inhibitor, increased Km

PFK is autoregulated by [ATP]

F1,6BP – F6P in gluconeogenesis is activated by inhibitors of glycolysis – reciprocal regulation

controlling futile cycle. If you have both enzymes active at the same time then wasting energy!

Exception to this is when you are GENERATING HEAT!

PEP to Pyruvate via PK regulated by F1,6BP activator (feed forwarding stimulation, if we have lots of S

coming through then pathway will be ready to accept the high flux). Pyruvate can eb transaminated to

alanine, if there is alanine build up, it means we have lots of biosynthetic intermediate! STOP

PK = F1,6BP (activator), ATP (inhibitor), Alanine (inhibitor)

F2,6 BP and PFK2 Regulation of Glycolysis:

F6P to F2,6BP via PFK2 uses ATP



Reversible, F2,6BPase change F2,6BP to F6P which can go through PFK1 to become F1,6BP

F2,6BPase activators PFk1, and inhibitors F1,6BPase (a gluconeogenic enzyme)

F2,6BP one of the most powerful allosteric modulators known, build of S will activate the PFK1 because

some are donverted to F2,6BP

Key point: the product produced by PFK2 is activator of PFK1, buildup of substrate F6P) will cause an

activation of PFK1 and stops the gluconeogentic eneyzme to stop futile cycles.

Covalent Regulation of PFK2 and PK:

- These effects have to be rapidly activated and reversible, by adding a phosphorus.

- Ser/Thr/Tyr-OH (protein substrate)

- Using a kinase (Activate or disactivate)

- Using a phosphatase or a phosophoprotein (Activate or deactivate)

F2, 6BPase and PFK2 both exist on the same enzyme! How? Covalent modification!

When insulin activated (high glucose levels) – protein phosphatase activates PFK-2 to drive glycolysis.

When Glucagon activated (low glucose levels) – a kinase activates F2,6Bpase to stop inhibition of

gluconeogenesis.

This is a bifunctional enzyme

PFK1 and FBP1 are not = each are phosphorylated on its own. Two separate enzymes.

Regulation of Pyruvate Kinase (PK)

- Regulated by phosphorylation, last commited step of glycolysis, exactly what we want during a

hypometabolic state, saving glycogens storages to live longer under anoxic situations.- Phosphorylation of PK inactivated the enzyme. Dephosphorylated PK is much more active.

- The same for PFK1 and PFk2 (phosphorylation reduces the activity)

cGMP + PKG – phosphorylates PKK to PKK-P.

This PKK-P phosphorylates Pyr-Kinase to Pyr-Kinase-P which inhibits it. Non-inhibited Pyr-Kinase can

change PEP to Pyr driving glycolysis.



Note that PK-kinase phosphtase can dephosphorylate to inactivate the PKK and stop the inhibition of

Pyr-Kinase.

PK is also regulated by allosteric modification – feed fwd of F1,6BP product of PFK1, these 2 effect

happen at the same time!

Vmax = maximum reaction rate of enzyme

Km = S0.5 = [substrate] that results in ½ Vmax

Ka = [activator] that increases rxn rate by 50%

I50 = Ki = [inhibitor] that decreases rxn rate by 50%.

If you require high concentration of activator or inhibitor to reach Km, it means the enzyme is less

sensitive to either the inhibitor or activator.

Phosphorylation of PFK1, PFk2, PK is what we want in hypometabolic state to stop the flow through

glycolysis in tolerant animals!

PK in anoxic, reduced sensitivity to Ka and increased sensitivity to Ki. Reversed Pasteur effect.

Metabolic Arrest and Evidence of Allosteric and Covalent modification:

Turning on/off glycolysis vs. gluconeogenesis phosphorylation just like PFK2/F2-6, and PK, PFK reduces

the activity. Remember, tandem enzyme which activates the phosphatase as well.

Turtles have only one mechanism to produce ATP in anoxia, lactate as alternate end product. But the

shell serves as a buffer and allows them to maintain the hypometabolic state for a long time.

The brain and red muscle have used a lot of glucose producing a l t of lactate, liver and heart don’t

produce much lactate, which says the tissues respond to anoxia at different rates, brain and red muscle

respond slower, still breaking down glycogen, you have to maintain the activity of brain for all

organisms!



Red muscle has to turn off all the oxidative enzymes and turn on the glycoltic enzymes and then reverse

that with time. With the rate of increase of lactate you have reduction in production of lactate overall in

liver, but in hypoxic state you have increase bc liver is responsible for gluconeogenesis to supply glucose

to other tissues.

White muscle is glycolytic muscle, best activity.

For PK, after 1 hr you have reduction of substrate, increase in production: stimulation of the enzyme,

activation of PK in all tissues, but after 5 hr, PK now have an increasein [S] and reduction in [Pyr],

enzyme must have been inhibited, this data suggests that PK is the most important enzyme in transition

from hypoxia to anoxia.

F2,6BP levels in Turtle organs and PFK-2 activities in whelk organs.

Activator of PFK1, in a hypometabolic state you expect a sig reduction of the allosteric activator,

because you want to turn off the enzyme.

Whelk dropped significantly. While turtle increased ? This increase may not mater because the enzyme

itself is less sensitive to activator during anoxia.

PFK2 and PK both show reduced enzymatic activity in anoxic. F2,6BP also reduced activity. PFK1 is

somehow controlled by the reduced activity of PFK2.

This is again to redeuce the flux through glycolysis, reverse Pasteur effect for anoxia tolerant organism.

PK and PFK ultimately regulated by ATP and Energy status of the cell:

- Enzymes when phosphorylated become more responsive to adenylein levels in the cell.

-

The energy status of the cell has a large effect.- If you have lots of citrate lets turn off those enzymes, inhibitor.

- IN anoxia, you increase the sensitivity to inhibitors so you require less citrate to inhibit these

enzymes (increased sensitivity)

PK is the major glycolytic control point in the transition to anoxia in anoxia tolerant organisms.



Based on the kinetics (Km) glycolysis is generally very sensitive to changes in ATP, ADP and AMP during

anoxia.

Therefore ATP demand can auto-reulate ATP supply.

Second smessenger medated regulation of PFK2

Glucagon (too little glucose) increased cAMP which activates the protein kinase to activate the FBPase-2

activity to inhibit glycolysis and stimulate gluconeogenesis.

Insulin (too muhch glucose) activates protein phosphtase, activating PFK2 stimulates glycolysis and

inhibits glucoenogeisis

PK

- Responsible for entry in anoxic state, activated by PKG, GTP producing cyclic GMP which is going

to activate PKG. PKG will activate pyruvate kinase kinase that phosphorylates PK, reducing its

activity (reducing

In liver this doesn’t happen, its producing glycogen for all organs. This happens in all other organs to

turn off PK.

In estivating snails, it takes 10 minutes for them to recover from anoxic states. Very rapid

phosphorylation, covalent modification. But don’t forget you have to control all pathways that produce

or use ATP to go into hypometabolic state.

Glucagon (low glucose) triggers cAMP from AC, cAMP binds to PKA to activate its catalytic domain which

turns off glycogen synthesis and activates glycogen phosphorylase to change glycogen to glucose.

Measuring Metabolic Arrest:

O2 Regulator vs. O2 Conformer!



- The other option is to be an O2 conformer, even if you have high O2 avaibility these O2 will

reduce their metabolic rate, conforming to available O2 in environment –this is usually

correlated with metabolic rate.

O2 regulator :

- Maintain oxygen usage during low PO2

- Resist a change in O2 change over a large range. Physiological adjustments that allow the

change, most show a critical PO2 at which point the O2 usage drops off very significantly. They

can no longer survive below that level.

- Oxygen consumption decreases rapidly once some critical point ~40 Torr and stops even when

some O2 is present (because they can’t reduce their metabolic rate enough to be able to

provide adequate ATP).

- ORGANISM CANNOT SURVIVE ANOXIA>

- Tachycardia and hyperventilation at low PO2.

O2 conformer:

- Low o2 usage but producing anaerobic end products at this point on the curve, still produce

some ATP by oxidative phosphorylation but not enough.

- Although they drop O2 usage, they are fine as they have enough O2 to survive, so easy to

determine Pcrit for O2 regulator and hard to do so for O2 conformer (they don’t all survive in

anoxia, most that do survive in anoxia are oxygen conformers).

- Many conformers use anaeboric end product formation to continue living and generate ATP.

- Utilization of anaerobic pathways has to go with metabolic rate depression.

Measuring Metabolic Arrest:

Need a measure of metabolic rate – under aerobic conditions we can measure:

O2 consumption ATP/O2 ratio is 30/6 or 5.

Measuring Anoxic Metabolic Arrest:

Under anaerobic conditions we can measure lactate

3 ATP from glycogen or 2 ATP from Glucose.

ATP/Lac = 1 or 1.5 for glycogen

What if lac is not the direct end product?



Direct and indirect calorimetry:

Direct calorimetry – measure heat of entire organism tissue or cell.

Indirect calorimetry –take end product such as lactate and glucose, measure heat generated.

Measure lactate produced in organism then multiply it by the heat produced by combustion of

lactate. It gives you an estimate, compare it to direct calorimetry results. If they are close then lactate

is the primary product.

In turtles, 60% of heat can account for lactate, 40% they don’t know!

Can determine how much heat produced of combustion of lactate rcorresponds to how much heat is

produced by an organism as it is metabolizing!

Lac production = ATP product.

Anerobic lactate production or ATP turnover, given as a rate lactate produced/umol*g cells

Indirect calorimetry – bomb adiabatic calorimeter –used to determine caloric equivalents. Adiabatic –

any heat made by the combusting molecule will go back to combust the molecule no heat lost to the

vessel. Not true however there is a calculation you can use to remove that inaccuracy.

Large room-size calorimeter used to measure daily caloric requiremnts or energy expenditure during

exercise.

Flow microcalorimeter – direct calirometry

Uses:

-

Measure heat generated in biological systems- Chemists to measure enthalpy changes in chemical rxns

- Pharmacologists to measure time dependent decomposition of drugs

Heat flows from the sample to the bath.



The flask is producing heat, the organism is under a controlled situation and we measure the heat

released from the organism –direct calorimetry. You haven’t combusted the material completely like the

bomb.

Can control conditions like anoxic or normoxic etc.

Lactate (glucose) anoxic combustion: Caloric equivalent, heat per mole produced from the formation of

a known end product – lactate = -55 kJ/mol

Have to take into account that H+ binding neutralization generates heat!

Turtle inside a flow calorimeter vessel:

- The rod holds the turtle’s head in the water, bubble nitrogen to make it anoxic, and after a

certain amount of time can then measure the end products produced from tissues.

This is a direct measurement of metabolic rate.

Kill turtles to get concentration of end products. Calorimeter measures heat.

Turtle is a oxy conformer – as soon as the O2 concentration is reduced it reduces its metabolic rate and

heat production. They don’t have physiological mechanism to regulate the O2, deloveped cell and

tissues to put in the calorimeter to avoid using the whole turtle.

Heat loss is decreased by 85% and blood oxygen levels similarly.

Normoxia maintains regular heat loss, when anoxia kicks in there is a clear reduction of metabolic rate.

Used slices of brain to put into flow calorimeter.

Cyanide, inhibitor of cytox is somewhat mirriors whats happening in the anoxia state. Reducing the

movement through oxidative phosphorylation. Much more rapid drop.

CN knocks out the mito oxidative phosphorylation.



When you add CN, it reduces the rate of heat production close to anoxic levels of heat production. They

found that CN didn’t seem to reduce the rate of heat flux the same as anoxia, in brain cells it did but in

hepatocytes it didn’t They came up with a couple of suggestsions:

- Some oxidative enzymes that use O2 as a substrate such as a peroxisomal catalases which could

contribute to the higher heat production.

The spikes are movement of org seems to be movement everytime they change O2 levels,with reduction

o the O2 rclear reduction in movement. Reduced heat flow through calorimeter.

Pcrit fails somewhere between 8.66 and 2.66, see a significant drop of phosphoargnine, significant

increase in strombine alanopine etc. can guess that oxidative phosphor can no long contribute enough

energy and alternative end products kick in.

In the indirect calroimetry seems to account for the direct calorimetry.

Example calculation of calorimetry:

From the rate of lactate production in anoxic hepatocytes a rate of heat production can be calculated.

6.5 umol of lactate / g * hr

Caloric equivalent for lactate is -55 kJmol/lactate

Figure out heat produced:

6.5 umol*lactate/g*hr x -55 kJ*mol/lactate

0.0000065 mol of lactate/g*hr x -55 kJ/mol of lactate

= - 0.0003575 kJ/g*hr (heat of production of lactate)

Since 1 W = 1 J/sec

- 0.3575 J /g*hr x 1 hr / 3600 seconds = -0.000099 J/g*s = -0.000099 W/g*s



Measured heat flux in hepatocyte is -0.23 mW/g*s

-0.099 mW/g*s / -0.23 mW/g*s = 43% accounted for by lactate

Therefore, there is missing heat. Is there an alternate end product?

One also has to consider the acid (H+) produced by anoxic metabolism. Each time the lactate is

produced there is an ATP produced and H+ is produced due to ATP hydrolysis (heat of neutralization).

Mix acid and base together and heat is produced, same for anoxic cells!

For hepatocytes study, HEPES buffer was used as support saline.

Heat of neutralization = -21.7 kJ/mol of H

1 H+ is produced per ATP, and from glucose the production of 1 lactate is 1 ATP.

So -55 kJ/mol of lactate + -21.7 kJ/mol of H+ per Lac

= -76.7 kJ/mol of lactate

6.5 umol of lactate x -76.7 kJ/mol of lactate

-0.138 mW/g

-0.138/0.23 x 100 = 60%

Still 40% of heat is unaccounted for.

This is the exothermic gap, some anaerobic end product that is not measured probably. But the other

explaination may have to do with dying cells, production of heat via apoptotic pathway. So still in most

anoxia tolerant organisms they don’t know what this is.



Signal Transduction Pathways Involved in Metabolic Arrest

Adenosine is very potent metabolic regulator –neurotransmitter, promoting sleep, hypometabolic state,

hibernation.

Normal blood concentration is 2-20 uM

- It’s a cardioprotectant, reduces heart rate, vasodilation, limits the venus return to heart, used in

cardiac bypass surgery, reducing cardiac output.

Very potent vasodilator!

Several different adenosine receptors with different sensitivies to adenosine.

A1 receptor neuronal (inhibitory receptor)

A2A receptor neuronal but mainly smooth muscle

A2B receptor and artieral endothelium (vasodilation ctrl)

A3 kindyes (not a high affinity)

Depending on the situation, stress, these receptors can be active or inactive.

Many of these physiological effects are consistnet with anoxia tolerance but much are done on anoxia

intolerant organisms. This is exactly what an organism would want to do in hypometabolic state.

Adenosine is present in all cells and can effect all system as we want in hypometabolic state.

Bradcardia, bronchodilatoon, stimulation of gluconeogenesis

Channel arrest!

ATP utilization pathways have to equal the ATP production pathway, so to maintain an ATP level you

have to keep them the same. So when an organism gets into a stressful state, the producing pathways



slow down and the utilization keeps going at first. This is a good way for the organism to REALIZE there is

a stress. Later must decrease utilizaiton and production equally.

1000 fold difference in concentration of ATP and adenosine.

10K fold different between Ca extracellularily and intracellular.

Which funcitons in same manner.

So in anoxia, excessive break down on ATP

ATP-.ADP-.AMP-.Adenosine-.Inosine

Adenosine is easier to measure because small change in ATP leads to large change in adenosine.

1% decrease in ATP = 0.03 mM results in a 10 fold increase in adenosine.

Pathways of Adenosine Formation

LIVER CELL -

AK = adenosine kinase can re-phosphorylate adenosine to AMP, during normoxic state, this is a very

active enzyme. It is believed that this cycle keeps the cell prepared to response to chanes.

Tissue tend to maintain a low level of activity so that it can respond to changes quickly. IN the anoxic

state, AK is inhibited (0 activity) so adenosine increases dramatically, as it can`t be converted to AMP.

Adenosine is then transported out via the equilitive transporter (ENT) or the the sodium linked

transporters.

THESE CHANNELS ARE NOT TOO ACTIVE IN SYSTEMS WE ARE TALKING ABOUT.



The MAIN POINT:

- AK is inhibited in anoxia meaning that adenosine can build up and leave the cell and bind

outside and stimulate the pathways outside the cell.

ATP very short lived outside the cell, 200 ms half life.

cAMP also contributes to adenosine formation, cAMP -. AMP via PDE

AMP – Adenosine via 5`nucleotidase!

In anoxia, it exits via ENT because it moves down its EC gradient, concentrative nucleoside transporter

(CNT) allows adenosine to travel back if there is too much EC adenosine along with Na.

Adenosine Receptors are G protein effectors (trimetric G-protein)

Can function as GEFs, once adenosine binds in changes conformation and turns into GEF stimulates the

alpha subunit to release the GDP and bind the GTP. Alpha subunit then diffuses away and can activateother things, the beta gama subunit as a dimer can also activate many things. Doesn`t dissociate, stays

bound to the receptor. The system has GTPase activity and will eventually hydrolyze the GTp to GDP.

This system can be stimulated or inhibited in many ways.

TARGETS:

A1 receptor – inhibitory, activation by A1 adenosine leads to an inhibitory response, LESS ACTIVATION

OF PROTEIN KINASE A LESS CAMP LESS INHIBITION OF GLYCOGEN SYNTHESIS. Inhibition of channels,

and PLC.

A2 receptor – stimulatory, Stimulates AC and PLC. PIP3 and DAG production.

Heterotrimetric G protein is a local effector, it can activate Guanlyate cyclase (GC) which takes GTP and

makes into cGMP.

cGMP is then used to activate PKG which activates PKK, which phosphorylates PK and makes it INACTIVE

(hypometabolic state). It promotes gluconeogenesis! Reduces flux of glucose into glycolysis.

Opening of Ca2+ channel (as in A2 receptor) can also stimulate guanylate cyclase through a calmodulin

effector.

Three forms of GC linked proteins:

1. Hormone receptor integral membrane



a. Binding of hormone has direct effect

2. GC peripheral membrane protein

a. Can be activated by alpha subunit of heterotrimeric g protein or Ca through calM

3. Soluble guanylate protein

a. NO synthase is stimulated by the increase in concentration of Ca, NO then activates a

heme-based protein which can bind O2 this can then respond to lack of O2.b. Potential protein receptor which could respond to lack of O2.

Four primary downtstrema targets of adenosine-receptor activation – Ser/Thr kinases.

PKA – cAMP dependent

PKC – Ca2+ calmodulin dependent

PKG – cGMP dependent PK

PKC

DAG

– phospholipid dependent PK (DAG)

Kinase Phosphorylation Sites:

Serine – COH

Threonine – COH

Tyrosine – COH

There are many associated ser/thr/tyr phosphatases as well!

Change in Intracellular Calcium Levels opens the the voltage-gated L typed Ca channel up!

Ca then binds to CalM releasing regulatory subunit from catalytic subunit giving an cative Ca2+ lM kinase

which can phosphorylate other proteins, Ca can also affect other proteins like troponin C in skeletal

muscle

Ca can affect periphery bound guanylate cyclase to make cGMP as well.

PLC Response:



So PLC is activated by Galpha and cleaves PIP2 into IP3 and DAG. DAG then activates PKCDAG

which can

phosphorylate proteins. More importantly, DAG is cleaved into Arachidonic acid, which is a precursor for

eicosecnoids, this is prostaglobadin –invovled in pain managene.t

Ip3 goes to open Ca channels in the ER or SR (in muscle) and Ca2+ is released, this causes PKC to moveto the membrane where DAG activates it.

IP3 is short liked so sometimes it can be phosphorylated into IP4 so it lasts LONGER in the cytoplasm.

Ca2+ released can also bind to CalM which releases the regulatory subunit making a Cacalm kinase.

AC Response:

AC activated by Gs or Inhibited by Gi

When activated AC converts ATP to cAMP, which binds to PKA making it catalytic. This PKA can then

phosphorylate proteins like PFK1 which can inhibit its function.

When ATP low, in anoxic tolerant organisms reverse Pasteur, reduce flux.

Multiple actions from a single EC signal!

Amplifing enzyme =- PLC and GC and AC

Some crossover between paathways.

Ion Channel Arrest and Regulation of ATP Demand

- Study other orgs to transfer knowledge got mammalian cells. Especially during surgery,embryonic cells can be somewhat hypoxic tolerant however not as good as turtle.

What do we know about anoxia tolerant turtles that can suppor the concept of ion channel arrest?

What occurs in a turtle during 6 mo of anoxia, in a N2 atmosphere or submerged at 3 degrees:



Whole animal:

- BMR decreases by 90% basal metabolic rate!

- HR decreases from 30 to 0.4 bpm – reduce metabolic rate so as not to require too much blood

flow- EEG decreases 80% - brain activity, shutting down neurons

- Oxidative metabolism inhibited

- ATP Turnover sustained by anaerobic metabolism (glycolysis) – supplemtsn ATP formation for a

time, but can’t sustain it for a long period of time during anoxia, some point there has to be

metabolic arrest.

- Animal appears comatose, awakening periodically to explore surroundings: therefore, muscle

activity severely reduced (less ATP demand).

Blood:

- Lactate increases from 150 to 200 mM –turtle has buffering system (shell) and still there is an

increase!

- pH drops from 8 to 6.8, for a time it was stable but in time it drops.

- Ca2+ increases from 5.5 to 36 mM

- Mg2+ increase from 3.2 to 22.4 mM

Presumably it balances the electrical charge of lactate. COUNTER ION BALANACE.

Cell hepatocyte:

- Na+/K+ Atpase activity decreases by 70%

- Membrane voltage is UNCHANGED.

- NMDA receptor – NMDA glutamate activated ca channel, ca is cytotoxic and can bind to

proteases and peptidases can cause cell death.

- Whole cell ion conductance decreases by 60%

A stragey to survive anoxia is to decrease tissue activity.

Heart – reduce contraction and pumping

Brain – reduce electrical activity (SPIKE ARREST) – change in ion channels lead to a reduction in th e# of

action potential produced or harder to produce (threshold increased?

Muscle – reduce muscle activity

This is evidence of metabolic arrest, but how is it achieved?



- A universal mech capable of reducing metabolic activity in all tissue may be an acute decrease in

plasma membrane ion channel activity. ION CHANNEL ARREST.

Cellular data support his.

Further support, Ca2+ and Na+ channel blocks significantly improve organ transplant outcomes. This is

seen as a protection for the lack of oxygen in organs.

In pleak channels/k channels responsible for determining membrane potential continually k is flowing

out atpases has to pump k in contiously outflow could be reduced.

If you have metabolic arrest, you stop atp production which means you also must reduce ATP utilization.But, if there is a leak then you have to use ATP to fire ATPases to maintain membrane potentials. So if

you can reduce leakage via channel arrest, then you can maintain a low ATP turnover.

Channel Arrest Hypothesis:

1. Stress tolerant organisms outhg tot have fewer ion chnanale per area membrane than intolerant

organisms

a. Fewer doesn’t me reduction of flux. Some channels may have higher flux.

2. Stress tolerant organisms ought to have mechanisms by which ion channel permability can be

regulated.a. Specifically down regulate or movement into a hypometabolic state.

b. Have to be able to come back so upregulation is also required.

EVIDENCE:

- Ectothermic organisms DO have fewer ion channels per membrane area, about 5 x less.

- However, fewer ion chnnaels is not enough to account for the observed stress-mediated

decrease observed in anoxia-tolerant organism.

- Therefore the second prediction must also hold.

ATP demand by Na+K+ atpase is LARGE!

- Na+ K+ atpase is the primary pump for maintaining cellular Ec gradient and membrane voltage.

o In brain this pump can consume as much as 75% of total ATP, in liver 30%

o Theefore, reduced ion leaage = reduced pump acitivty = ATP savings!

Examples of Ion Channel Arrest:



1. K+ channel and adenosine, adenosine neurotransmitter, reduction in ATP produces large

increase in adenosine. Released outside cell and attached to receptors that affect K channels.

2. Na+/K+ atpase

3. [ATP] and membrane potential, ATP concentration remains neutral in normoxia and anoxia, cells

are active without membrane potential cells die.

4. Na2+ channel5. Ca2+ channel –the NMDA receptor

6. AMPA and GABA receptor – Cl channel and inhibitory neutoransmitter GABA.

Adenosine in Anoxic Turtle Brain:

- Increase consistent with 30 min time of metabolic depression seen in calorimetry exp.

- 1% decrease in ATP causes 10 fold increase in adenosine!

- Continued rise in adenosine, organism still trying to keep up normix metabolic rate, but they

can’t keep up so theirs is a drop in ATP.

ATP sensitive K+ channel, opens when ATP decreases hyperpolarizes cell.

K is an indicator of cell injury any stress will release k to Ec space, can be measured and is an indicator of

breaking of membrane integrity.

K is just one measure of cell survival because it is easy. Turtles and crucian carp can maintain k in cell

and maintain cell integiry.

Adenosine receptor antagonists promote K+ release

In turtle brain, adenosine receptor blockers promote K+ release.

This is not seen during normoxia (no lack of ATP, K+ ion not stimulated to open).

EC k rises when adenosine receptors are blocked, adenosine binding to its receptors leads to reduction

of open k channels or release of K.

Conclusion of adenosine exp:

Release of cellular K is a hallmark of anoxic injury in anoxia INTOLERANT species.



Does not occur in anoxia tolerant species like the turte and carp.

But if adenosine receptors are blocked, K+ release occurs in anoxia tolerant species.

Therefore, adenosine acting through adenosine receptors plays a role in the metabolic depression (or K+

ion channel arrest).

Na+/K+ ATPase

Rb can subtitiute for K 2K moves in per atpase hydrolyzed so 2 Rb would also move per ATP hydrolyzed.

Problem in measuring Na because of Na leak channels. So they used ouabain to inhibit the Na/K tapase.

Measure total Rb uptake – total rb + oubain = specific uptake.

Rb uptake total = all channels including NaK atpase

Rb + oubain = all channels except NaK atpase

The difference = NaK Atpase Rb intake!

Slope of Rb over time = rate of Rb uptake, if you divide by 2 you get the rate of ATP use because 1 Atp

= 2Rb

Result requires 4 exp’s

Normoxic = total rb, total rb with outabin

Anoxic = same two

Significantly reduced ATP hydrolyzed, 75%! Clearly shows there is a reduction under anoxia!

Adenylate Levels and Metabolic Rate in Hepatocytes



- ATP, ADP, and AMP maintained concentration, not much of a change. Together with the slide

before, concurrent drop in ATP utilization and ATP production.

Temperature strongly reduces metabolic rate. KCN doesn’t provide complete inhibition!

Normoxic and Anoxic PM Potential in Turtle Liver cells

- Voltage of membrane doesn’t change

- Very hard to depolarize, most cells you can just put ;ots of K and it would depolarize but had to

use valinomycin specific for K that allows K to move into cells

- NO DEPOLARIZING DURING ANOXIA!

Proportion of Total Cellular ATP turnover utilized by NaK atpase under normoxic and anoxic conditions

- 90% reduction in the total ATP usage

- But in the 10% of ATP, 74% is used by ATPase.

Important to maintain this to some degree in order to maintain membrane potential!

Rationale for Channel Arrest:

1. ATPase activity decreases by 75%

2. Membrane potential doesn’t change

3. ATP production decreases by 90%

4. No change in ATP concentration because of turnover staying the same

5. Decrease pump activity should result in decrease of Vm but it does not therefore membrane

permability to ions must be decreased (via one of the channels or all of K, Ca and Na)



Key points:

- Decreased o2 consumption

- Early increase in glycolytic rate

- Later decrease in glycolytic rate

- ATP turnover is maintain at a lower level in the absence of O2.

Scope for metabolic activity:

- Brain is 2^ of body mass but utilizes 20% of total O2 consumption – intelligence comes at a

price!

Evidence of Channel Arrest from Ca2+ and Na+ Channels:

Neuronal Ca2+ channel and excitotoxic cell death (ECD)

General scheme:

- [ATP] decreases, causes metabolic stress

- Pump failure (requires ATP) due to lack fo ATP, compromise NaK and Ca2+ Atpases

- Subsequent movmenet of ions and loss of ion gradients across PM this depolarizes the cell

- Following loss of ion gradients, comes massive reelase of neurotransmitter like glutamate.



In anoxia tolerant org, increase in Ca2+ during anoxia. Strage because Ca increase is linked to cell death,

but found that you do need an elevation and continue to maintain this concentration during anoxia. Not

a massive increase, just a slight one.

Continued:

How is ECD prevent in stress tolerant species?

- Process is reversible until cytosolic Ca increases beyond a certain level

- What is the pathway for calcium entry?

- In brain, Ca2+ unders through n-methyl-daaspartate receptors NMDAR; which is a glutamate

receptor subtype.

IN rat model mammal, NMDAR blocks are NEUROPROTECTIVE.

Ca2+ flows down NMDA receptors when they are activated, they do block these channels!

NMDA at resting membrane potential is blocked by Mg, in a depolarization Mg will be pushed out of

channel and lead to opening of channel.

This in addition to binding of the Glutamate ligand. The binding doesn’t allow for opening of the channel

alone. You need depol + binding.

AMPA, Glut ligand binding causes Na moves down its EC gradient, reducing the membrane potential (-

30 mV all positive charges decrease potential), depol = removal of Mg, which results in activation of

NMDA.

Ionotropic channel = receptor is an ion channel

NMDA is a coincidence detector – bc Mg blocks this channel you have to removal the Mg via depol

before Ca can rush in the cell.

At resting membrane potential of -60to -70 mV, Mg2+ blocks the passage of Ca2+ through NMDA.



This is a electronphoretic interaction, that is reversed when cell is depoll by Na+ entry via AMPA.

It is a conincidence detector since it may require many presynaptic inputs to supply sufficient Glut to

activate a sufficient number of AMPA receptors to sufficiently depol the cell to remove Mg2+ of NMDA

receptor.

Voltage-gated Na+ channel allows Na influx following cellular depol by Ca2+, Brevetoxin binds to these

Na+ channels can be used to determine the number of channels.

Can label brevetoxin and see our Na channels.

Anoxia Sensitive Cell Death:

1. Reduction in O2, proton gradient at mito is decreased and Ca is released from the mito

2. ATP falls due to no oxidiative phosphorylation

3. Na/K pump can’t function

4. Na flows down the Ec gradient

5. Reduction in membrane potential causes the release of neurotransmitters like Glu

6. Glu then binds to NMDA or AMPA or ACPD and causes a massive inlux of Ca2+ leading to cell

death.

K/ATP channel in anoxia tolerant can inhibit the release of EC K.

This massive release of glut in the area and lead to massive influx of Ca in the periphery, in stroke you

have a focal region and an area effect by release of glut, as they increase cereral blood flow to those cell

to avoid cell death.

K+ out will hyperpolarize but Ca2+ and Na+ in overhwhelms and depolarizes Vm.

Middle Cerebral Artery Occlusion Model (MCAO)

- A model to study hypoxia tolerance in mammalian brain and to study hypoxic preconditioning

(important in transplant and surgery)



The suture shown creates an ischemic region in the right hemisphere of the brain

The left side can be used as a contralateral control.

NMDA receptors blockers and/or adenosine (inhibitory NT) reduce tissue damage in the ischemic region.

Hypoxic Preconditioning

3 minute suture -------------- no damage

6 minute suture ------------- extensive damage

3 minute suture -------- 1 day recoveyr - 6 minute suture -- no damage

Cells have been preconditioned during the 3 min condition, great for surgery and transplant even in an

anoxia intolerant cell.

Demonstrates that even mammalian brain has some capacity for neuronal protection.

Glutamata Receptors in Anoxia Tolerant Animals

- The two glut receptors NMDA and MPA are also ion channels.

NMDA is responsible for large influx of Ca2+ needed to initiate an action potential and during ECD.

AMPA is used to allow Na+ into the cell acausing a depol sufficient to remove Mg block off the NMDA

receptor.

How do anoxia=toeraltn animals prevent ECD?

Micropeptide over a single channel, and measure the voltage from the signal channel, may not be

represent of the whole community of chnnales or just apply NMDA to whole cell and measure as

community.



Measuring the open probability of NMDA channel, adenosine will reduce the open probably of the

NMDA receptors, inhibitory protective NT, in an anoxic situation yuou have the same reduction in the

open probabily of the NMDA receptors. 8PT is an inhibitor of the A1 inhibitory adenosine receptor

and you no longer get the reduction in the open probability of the NMDA channels.

Increase in current by addition of NMDA in normoxic for about 40 minutes.

Anoxic or adenosine application, after 40 minutes the major reduction in current flow in the whole

cell. Same thing happens with anoxia.

Ca is involved in current change of NMDAR, if you add BAPTA (Ca2+ chelator binds Ca2+) there is a an

increase in the channel current. Means that Ca2+ is playing a role in anoxia depression of NMDAR.

Adenosine receptors modify the NMDA receptors as they have G proteins that can activate the AC.

AC makes cAMP which activates PKDA which phosphorylates NMDA.

Calyculin A can block the dephosphorylation of NMDA by PKA. Dephos is required for the reduction of

NMDA. They thought that phosphorylation by PKA was the key, but dephosphorylation was required.

Whole-cell AMPA receptor currents

AMPA currents inhibited by anoxia. IN anoxia the AMPA receptor was reduced by 60%, they lead to

the depol that leads to removal of mg from NMDA receptors.

GABA receptors can function as a calmp or shunt to prevent an action potential.



GABA is a primary inhibitory NT, GABAa

receptors are ionotropic as well, post-synaptic. GABAb is

metatotropic, involved in reduction of presynaptic glut much high[Cl] outside the cell.

GABA usually hyperpolarizes (flux of Cl-) cells Reducing ECD.

[GABA] increases dramatically in anoxic turtle brain, general anesthietcs are functioning by inhibiting

NMDA and exciting GABA.

- Some anoxia-tolerant organisms do this naturally, and could represent a natural anesthetic

mechanism

GABAergic “shunting” inhibition

Neonatal mammals: GABA is stimulatory NT, CL is high inside and will excite the cell, the membrane

potential will move towards the reversal potential of the Cl, they have more Na/Cl co transporter in

neonates which is why they have high [Cl] in the cell.

Hyperpolarizing = inhibition

Depolarization / shunt inhibition, lots of GABA and its channels, prevents further depol no ECD.

Spike Arrest, may be a better term than Channel arrest in neuron cells

Significant reduction of AP frequency when anoxic.

Looking at the open probability of channels, in anoxia they are reduced, to reduce need for ATP, in

neurons although you see an increasei nwhole cell condustance during anoxia, all of which is entirely

due to the movement of Cl through AGABA, as GABA is released in huge concentrations in anoxia,

increases whole cell conductance. SO in terms of neurons this is called spike arrest. You aren’t blocking

channels, you are arresting Aps by preventing depol.



Opening a massive number of GABA channels, massive amount of Cl flow, protecting it from further

changes in membrane potential.

Na+ Channels Arrest

Voltage sensitive Na+ channels required for initiating the AP

Initiates AP, major current carrier during AP.

U can label Brevetoxin and locate these channels.

NOrmoxic brain, incubate brain lslice with H-brevetoxin 1 g brain measure how much of that channel is

bound to the membrane.

Set measured normoxic value to 100%, in anoxic brain there was a 58% decrease! Na channels have

been inactivated perhaps even phagocyized by the membrane!

1. Channels removed from PM (more likely than…)

2. Channel modified –phosphorylation irreversibly

Oxygen-Sensing Systems:

- If these organisms are responding to O2 changes, there has to be sensing systems.

1. Systemic – communication between tissues

2. Cellular –communication WITHIN cells

3. Molecular – transcpriotoinal response

Acute sensing in 1 and 2 , chronic response is 3.

If the organism isgoing to stay in anoxia for a long time better to have a transcriptional response to aid it.

Production of ROS as an O2 sensor, some say that increase production of ROS some say it’s a

reduction of ROS that leads to coping mechanisms…



1. Sysemtic O2 sensing

1. Carotid bodies (peripheral chemorecepotors)

a. Sense reductions in arterial oxygen tension

b. Located above carotid bifurcation

c. Stimulate respoiratory center to induce hyperventiliation

d. Currently being investigated

e. Start firing when O2 levels are below 60 this firing will change rates of resp.

located in the perfect spot for protection of brains’s O2 supply. Appear to be the

MOST IMPORTANT.2. Central chemoreceptors (respiratory center of the brain)

a. Sense pCO2 and maybe pO2 (or protons/decrease in pH)

b. Located ventral medulla

c. Mechanism linked to cellular acification (pH)

d. Either HCO3 or glycolytic production of lactate can all lead to pH

3. Pulmonary Neuroepithelial bodies

a. Neuro-secretory cells, located in bifurcaitons of the bronchi at the lungs.

b. Sense reductions in inspired oxygen partial pressures

c. Innervated clustered of amine + peptide containing cells

d. Widely distributed throughout airway mucosa of mammalian lungs

e. Mechanism being investigated

4. Adrenal Chromaffin Cells – can sense oxygen (adrenal gland can release epinephrine or

adrenaline)

All sensors above may involve – GLOMUS cells – specialized o2 sensing cells originating from enural

crest cells – can fire AP and release NT, can control and monitor ventilation, the initial binding of O2 to

them are still argued about.

Possible underlying mechs of Systemic and Cellular O2 Sensing:

- O2 sensing K+ channel

o Po2 decreases and channel activity decreases

o Less K+ leaks from cell

o Depol –more excitable

- NADPH oxidase

o PM sensor

o Produces ROS in proportion to Oxygen concentrations



- Mito free radical production

o IMM ubisemiquinone-bc1 complex produces superoxide byproduct in proportion to

ambient oxygen concentrations.

Model of O2 Sensing

a. Ligand Model

His was believe to be a heme based protein but doesn’t appear to be anymore.

Oxy heme deoxy heme effector and O2 sensing channel FOCUS

b. Redox models – sensing via superoxide (NADPH oxidase / SOD)

c. Proteins have thiol groups and cys, the absence of O2 leads to reduction of those thiol groups

into SH, most work is done with Ca release, it is not believed that this is the O2 sensor, but

instead just involved in the RESPONSE to O2.

NADPH redox model is common in neutorphils used to kill bacteria.



Systemic O2 sensors – carotid and aortic body chemoreceptors ,they are not pressure receptors like

baroreceptors. Can change the depth and rate of ventilation.

Cs+ blocks K channels

TTX blocks Na+ channels

Significant reduction in the K+ current with low O2 levels.

Under anoxia, increase in the rate of pace maker potential (SA) increase in the frequency of Aps.

Single ion channel measurements, measured the open probability of these channels. Significant

decrease in the open probability of these channels in low O2 situations.

Theory is that o2 is not directly involved (binding of O2 to channel) its general blelif is that they are

indirectly involved with a movable peripheral sensing molecule that move and open/close the channels.

Summary of Glomus Cell O2 Sensing



1. Blood vessel decrease in PO2

2. This interacts with a MAYBE heme based molecule (not sure)

3. K channels are CLOSED.

4. Depolarization of membrane

5. Activate voltage gated Ca channel L type, high flux. There also could be Na channels.

6. High intracellular Ca2+7. NT release

8. Activation of afferent fibers which send the info to CNS.

SOURCE OF CA2+ IS NOT INTRACELLULAR ITS EXTRACELLULAR ---- PROOF:

Possible Mito Mechanism:

If mito depolarizes due to lack of O2, Ca2+ will leak out causing vesicular fusion and release of NT.

Where does the Ca come from? EC and IC. In the absence of O2 you can have a breakdown of the

membrane potential in the mito that can lead to release of Ca. THIS WAS TEST WASN’T THEMECHANISM.

- An EC Ca2+ blocker was used there was no increase in intracellular Ca

- Ca2+ removed from bath saline there is no increase in intracellular Ca2+

- Conclusion: source of Ca2+ is extracellular

Cellular O2 Sensing

EPO is a model cellular O2 senosor:

- Glycoprotein hormone

- Stimulates RBC production in bone marrow (RBC have a limited life span constantly producing it,

and epo is relatively stable)

- Normally blood levels remain fairly constant.

- Becomes hypoxic – EPO levels increase and within hrs RBC count increases.

- The discovery of EPO led to therapies for patients suffering from reneal anemia (kidneys not

making EPO)

- Aplastic anemia – bone marrow RBC production insufficient

o Increase in EPO, increase in RBC count, increased viscosity, increased pressure on heart

and can cause stroke and death!

The EPO System involves HIF 1:

In NORMOXIA:



HIF-1alpha is hydroxylated by Prolyl hydroxylase, the HIF1a-OH can then accept a ligand called VHL, the

binding of VHL to HIF1alpha allows the ubiquitin to bind. This marks HIF1a for degradation in

proteasome.

InHYPOXIA:

Prolyl hydroxylase is an O2 sensing enzyme, it uses O2 as a substrate for the hydroxylation as well asFe2+ and alpha keto-glutorate

When all substrates are present it awill get one OH to HIF-alpha.

If HIF1a is not degraded it moves to the nucleus and dimerizes with HIF1B and this leads to the

transcription of EPO, LDH, VeGF – induce growth of vasculature.

From EPO research 4 conditions must be met:

1) During normoxia should be insensitive to metabolic poisons – CN (because O2

is still there, so heme sensor should still be able to sense it)

2) Co2+ and Ni2+ should mimic anoxic effects –these lock heme protein in deoxy

conformation – can’t bind O2.

3) CO should reverse anoxic effects – lock heme in oxy conformation (reverse of

cobalt)

4) Heme synthesis inhibitors should cancel effects of Co2+ and Ni2+

WRONG. This is due to prolyl enzymes and arginylyl and not heme proteins.

Molecular Oxygen Sensor HIF1

HIF is a protein that is a moeclular oxygen sensor.

After nuclear translocation in the asbsence of Oxygen. Asp hydroxylase can still hydroxylate the c-term

of HIF1a block its association with P300. Blocking transcriptional activation.

HIF1B is always present in absence of O2 the prolyl hydroxylase and asnn odesn’t function and H1F1a

can enter nucleus and dimerize with HIF1b.



HIF results in an increase of all glucose metabolism enzyme expression – PK, HK, PFK1 etc. Glycolysis!

HIF 1 has two rounds of gene expression that results in upregulation of different genes like the ones on

the western blot.

And glucagon can also activate 2ndary messengers which trigger CREB to bind to CRE which can also

downregulate genes like Krebs cycle enzymes etc.

Molecular Stress Sensor:

Environmental Factors can result in increase gene expression such as:

- High and low temperature

- Low O2

- Ethanol

- pH changes

- H2o2 and free radicals

- Viral infection

- Fever

- Heavy metals

- Salinity

- Exercise

Stress proteins are not just heat shock proteins!

Stress proteins – a general term for proteins that are transcribed and translated during or following

stress.

HSP and HIF1, hsps are proteins that stabilize the 3d structure of other proteins – chapearons that help

with folding or proteins into their 3d conformation, protects them from stress and respond after stress

helping them fold back or send them for degradation.



In unstressed cells HSP’s are CHAPERONES.

- Assist in protein folding from 1’ to 2’ structure

- Increase up to 1000 fold.

- However, when a stress occurs there can be a 1000 fold induction of a particular stress protein- Stress proteins are well conserved across species.

Classified into 6 major families based on molecular weight:

- 110 kD

- 90 kD

- 70 kD

- 60

- 40

- Small hsps – 10-20 kd

70 is the primary one in research!

Stress proteins confer thermotolerance and underlie pre-conditioning!

In ischemia if you can precondition the tissue we can protect in hypoxia intolerant tissues by turning

something on when it normally isn’t.

Survival rate of yeast after incubation at 37 degrees for 30 minutes before transfer into 50 degreesresulted in higher survival rate – correlating in expression of Hsp104! (Western densitometry)

Direct transfers all died at 10 minutes of 50 degrees, while the preconditioned animals lasted for 20

minutes at 50.

HSPs function as chaperones in unstressed cells – haperons after a stress can help refold, chaperones

require ATP to do this, expensive process but conserved as it is important for survival.

AA 1’ --- HSP refold - AA 2’ sequence -- heat shock --- denatured AA - Hsp70 use ATP to refold.

Despite being ubiquitous, the Hsp expressed differs between organisms.



Yeast expresses large amount of Hsp104, fruitflies mostly hsp70 while hsp 104 is almost absent.

Hsp 70 is the most common hsp: and is highly conserved, human hsp 70 is 73% identical to fruitfly!

HSF – heat shock transcription factor binds to HSE (heat shock sequence elemtn), activates transcription

of Hsp70 gene via RNAP2.

The regulatory model of transcriptional activation leading to de novo synthesis of HSPs:

IN normoxic conditions – a complex with at least 3 HSPs hold on to HSF so it can’t go to nucleus.

In hypoxic there is a dissociation of the complex, where the Hsps go help refold unfolded protein, and

the HSF1 monomers enter the nucleus and trimerize and become phosphorylated. This trimer of HSF1

can then bind HSE and activate transcription of more hsps.

Hsps that are produced can feedback and inhibit excessive transcription.

30 h dive and 1 h recovery dependent hsp72 expression in turtle

Very little change in early anoxia (0-12hr) in anoxia tolerant organism, not considered a stress. But after

12 hrs you get a significant increase! The liver had a sig reduction in the first 24 hrs of anoxia after

recovery, massive increase in all tissues including the liver, so in longer term anoxia, this is a stress, they

turn things on, will produce acidity long term lack of ATP.

2 stages of response to anoxia – 1. Defence shutting down the use of ATP. As no oxidative phos by

channel arrest. GABA transcriptional arrest, to reduce the usage of ATP because you can’t produce as

much, but later you have the rescue phase, turn on gene expression.

If you can get these changes before the anoxic event –then you have preconditioning!



Documents

Metabolic Arrest Cont