Neurochemistry 12011 Tim Murphy Objective: To understand the metabolic processes underlying the...
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- Slide 1
- Neurochemistry 12011 Tim Murphy Objective: To understand the
metabolic processes underlying the synthesis and metabolism of
amino acid and peptide neurotransmitters. Major points to be
covered: -regulation of metabolism by enzymes -metabolic processes
neurons share with other cells and organs -properties and functions
of enzymes and pumps (transporters). -metabolic contingencies
imposed by the existence of a blood-brain- barrier, i.e. the
central role of glucose -synthesis and metabolism of amino acid
transmitters and GABA. -glutamate -aspartate -glycine -neuropeptide
synthesis and the pathway to regulated release
- Slide 2
- Neuronal metabolism. Neurons share with other cells the need
and ability to synthesize nucleic acids, proteins, carbohydrates
and lipids. Likewise they share the metabolic processes required to
generate chemical energy for these processes: glycolysis,
pentose-phosphate shunt, citric acid cycle, oxidative
phosphorylation. Neurons must be able to synthesize and metabolize
neurotransmitters. Neurons must also synthesize second messenger
molecules needed to mediate signal transduction.
- Slide 3
- The brain makes use of general metabolism to find precursors
and in some cases the finished products for synaptic physiology.
glycine
- Slide 4
- Enzymes Help processes within neurons overcome activation
energy, and provide a site of regulation. Essentially all chemical
reactions in cells are mediated by enzyme, protein catalysts. A
catalyst acts by bringing together the reactants, and thereby
increasing the rate of a chemical reaction, without being
permanently changed in the reaction. Enzymes also allow the
coupling of energetically unfavourable reactions with reactions
that release free energy. If together the two reactions result in a
negative G, the coupled reaction can occur.
- Slide 5
- Enzymes lower activation energy for reactions. Mol. Biol. of
the Cell
- Slide 6
- Enzymes permit coupled reactions, for example falling rocks
turn wheel to raise water for a different type of work. Mol. Biol.
of the Cell
- Slide 7
- ATP is a useful energy currency since it can form high-energy
intermediates permitting the coupling of energetically unfavorable
reactions to favorable ones, shown is the amination of glutamate.
Mol. Biol. of the Cell
- Slide 8
- General Properties of Enzymes Enzymes are highly specific due
to the specific structure of the active site Substrate specificity
Reaction specificity Enzymes bind substrates in specific ways that
stabilize a reactive conformation, known as the TRANSITION STATE
Some enzymes require cofactors for complete activity (vitamin B6,
pyridoxyl deficiency can impact GABA synthesis).
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- Velocity (V) as a function of substrate (S) plot. Km Saturation
pseudo 1 st order
- Slide 10
- V=V max * [S]/([S]+K m ) With a competitive inhibitor, the K m
is increased but the V max is not effected. K m =K m *(1+[I]/K i ),
note when I= K i the K m doubles With a noncompetitive inhibitor
only the Vmax is reduced. V max =V max *(1-[I]/([I]+Ki)), note when
I= K i the V max halves Michaelis-Menton Equation, describes
saturable enzyme kinetics, also applicable to binding of ligands to
receptors. know this, it describes many interactions: enzymes,
receptors, protein-protein.
- Slide 11
- Km and Vmax The activity of enzymes can be discussed in terms
of their Km, a measure of the affinity of the enzyme for its
substrate, and the Vmax, which is the maximal velocity of the
enzymatic reaction. Km has two meanings: 1) the concentration of
substrate at which 1/2 the active sites on an enzyme are filled. 2)
the ratio of dissociation to association rates for enzyme substrate
interactions. Km=kdissoc/kassoc. Since the association rates of
many reactions at going the speed of diffusion, the strength of
binding and rates of reaction are often determined by the
dissociation rate. Although these terms are associated with enzymes
they are related to other saturable systems such as transporters
(Kt, Vmax) and receptors (Kd, Bmax).
- Slide 12
- Competitive inhibitors. Action: at the catalytic site, where it
competes with substrate for binding in a dynamic equilibrium- like
process. Inhibition is reversible by substrate. Effect: Vmax is
unchanged; Km, as defined by [S] required for 1/2 maximal activity,
is increased.
- Slide 13
- Noncompetitive inhibitors. Action:Binds E or ES complex other
than at the catalytic site. Substrate binding unaltered, but ESI
complex cannot form products. Inhibition cannot be reversed by
substrate.. Effect: Vmax is reduced; Km, as defined by [S] required
for 1/2 maximal activity, is unchanged. Knowing if something is
competitive or non- competitive is important since it determines
how much inhibitor you need relative to substrate (practical
implication!!)
- Slide 14
- Substrate or ligand concentration
- Slide 15
- Transport can be saturable.
- Slide 16
- Relative scales, simple diffusion rates will be low for polar
substances.
- Slide 17
- Channels and carriers.
- Slide 18
- Slide 19
- Since many transported compounds are charged their movement is
governed by electrical and chemical gradients just like small ions
such as K+, Na+, Cl-, and Ca2+.
- Slide 20
- Uniports-facilitative or uncoupled transport Molecules or ions
move down their concentration gradient via a specific carrier. In
contrast to a channel which will allow movement of thousands of
ions per millisecond and whose specificity is primarily mediated by
pore size, a facilitative carrier requires binding of a specific
substrate which induces conformational changes in the carrier
through which the substrate is moved, and then released, restoring
the carrier to its original conformation.
- Slide 21
- Carrier-Mediated Transport, Uniporters. Carrier types at the
blood brain barrier: hexose, monocarboxylic acid, large neutral
amino acid, basic amino acid, acidic amino acid, choline, purine,
and nucleoside carriers. These substances serve as building blocks
for all brain macromolecules and neurochemicals.
- Slide 22
- Slide 23
- Symports and antiports Couple movement of one molecule with
that of one or more other substrates. Energy is derived from
concentration gradients no ATP needed (directly) although
indirectly to establish gradient. The high-affinity pumps for amino
acids, and neurotransmitters are principally Na+-symporters, i.e.
the movement of Na+ down its electrochemical gradient provides the
free-energy required to move another substrate (neurotransmitter)
up its concentration gradient Na+/Ca++ antiporters, and Na+/H+
antiporters move these ions out of cells as Na+ enters.
- Slide 24
- Na+, Ca2+ exchange Glutamate protons
- Slide 25
- The Na+ gradient can be used to pump glucose uphill.
- Slide 26
- Slide 27
- Primary active transport Systems utilize the free-energy
obtained by ATP hydrolysis to move ions against concentration
gradients (uphill), i.e. Na+-, K+-ATPase or the Ca2+ ATPase.
Estimated to require up to half the brain ATP, while other
biochemical processes including protein, lipid and neurotransmitter
synthesis together use perhaps 10%. Other primary pumps, such as
Ca2+-ATPases and proton pumps probably account for the rest. The
brain uses 20% of total body oxygen consumption, thus 10% of total
is used primarily to maintain neuronal ionic gradients via this
pump.
- Slide 28
- Na+, K+ ATPase
- Slide 29
- Energy is directed into the pumping process by the
3Na+-dependent phosphorylation, followed by the 2K+-dependent
dephosphorylation. Phosphorylation induces a conformational change
that moves 3Na+ to the outside of the cell. Pump stoichiometry is
3/2 making it electrogenic.
- Slide 30
- Fundamental Neurosci. 2002 Zigmond et al.
- Slide 31
- Role of the pump in resting membrane potential. If pump is
blocked with ouabain (blocks binding of K+) an immediate small
depolarization occurs (only a few mV), however membrane will remain
relatively constant as it is largely determined by K+ permeability,
however the membrane is also slightly permeable to Na+ and over
time the membrane potential will depolarize if Na+ diffuses in
unchecked by the pump.
- Slide 32
- Glucose Is the major fuel of the brain because it is the only
fuel which enters in sufficient amounts to support the energy
requirements. Glucose gains access to brain and into cells by
specific carriers - blood levels much higher than brain levels,
thus glucose moves down its concentration gradient via facilitative
transport. Glucose utilization of tied to neuronal activity and
increased blood flow, basis of PET functional imaging with
2-deoxyglucose. Isolated neurons can use other fuels such as
pyruvate and lactate, but they normally are not BBB permeable.
- Slide 33
- Blood (~6 mM glucose). 4X Glut-1 expressed on the ab-lumenal
side Farrell and Pardridge 1991 CSF (~4 mM glucose). Fundamental
Neurosci. 2002 Zigmond et al.
- Slide 34
- Glucose transport The Km of the BBB glucose transporter is
about 7 mM, which is about the level of plasma glucose, thus brain
glucose varies directly with changes in blood levels. The blood
brain barrier transporter is Glut-1. Neurons possess a carrier of
higher affinity, Glut3 Km = 200 M, allowing them to extract glucose
from the extracellular space. Within neurons, glucose is
immediately phosphorylated to a charged, impermeant metabolite,
glucose-6- phosphate, thus the intracellular glucose concentration
is effectively zero. Why is it advantageous to reduce the apparent
free concentration of glucose.
- Slide 35
- Used in PET scanning. Fundamental Neurosci. 2002 Zigmond et
al.
- Slide 36
- Glycolysis and TCA cycle Within the cell, glucose enters the
glycolysis pathway in the cytoplasm, and via pyruvate and
acetyl-CoA, in the mitochondrial tri-carboxylic acid cycle (TCA) or
Krebs cycle. In these systems, reducing equivalents are generated
and via oxidative phosphorylation they generate ATP, the chemical
fuel for the brain. Glycolysis and the TCA cycle are also the
source of non-essential amino acid precursors used to synthesize
the neurotransmitters glutamate, aspartate, GABA, and glycine.
- Slide 37
- Slide 38
- Blood brain barrier. What is the blood brain barrier (BBB)? The
existence of a blood-brain-barrier prevents molecules in the
circulation from freely entering the brain. Prevents constant
fluctuations in circulating metabolites, ions, and hormones from
directly influencing neuronal activity. Diffusion allows passage of
gases, i.e. (O2 and CO2) and lipid soluble compounds, i.e.
psychoactive drugs.
- Slide 39
- The blood brain barrier largely occurs at capillaries through
astrocyte endfeet and endothelium tight junctions. Transport across
it is selective. Carrier types at the blood brain barrier:
hexose,monocarboxylic acid, large neutral amino acid, basic amino
acid, acidic amino acid, choline, purine, and nucleoside carriers.
Drewes LR. Adv Exp Med Biol. 1999;474:111-22.. Endothelium
- Slide 40
- Iadecola and Nedergaard 2007 Nat. Neurosci.
- Slide 41
- Perivascular glia contain high levels of the antioxidant
tripeptide glutathione Sun et al. 2006.
- Slide 42
- Paulson, European Neuropsychopharmacology 12, 2002, Pg. 495
Fig. 1. Characteristics of the endothelium. In the muscle capillary
(upper) there are pores or slits between the endothelial cells
allowing bulk flow of water and smaller solutes between the blood
and the extracellular space in the tissue. In contrast, the brain
endothelial cells (lower) are connected by tight junctions. No
pores or slits are present preventing bulk flow. Water therefore
has to cross the bloodbrain barrier by the mechanism of
diffusion.
- Slide 43
- Brain activity and blood supply are tightly linked. It has been
known for over 100 years increased neuronal activity is associated
with increases in blood flow. Roy CS, Sherrington CS (January
1890). "On the Regulation of the Blood-supply of the Brain". J. of
Physiol. 11 (1-2): 85158.17. Changes in blood flow or oxygenation
are used a surrogate measure of neuronal activity.
- Slide 44
- Glial and neuronal control of brain blood flow David Attwell1,
Alastair M. Buchan2, Serge Charpak3, Martin Lauritzen4, Brian A.
MacVicar5 & Eric A. Newman6 Nature 2010 468:231 Glial and
neuronal control of brain blood flow
- Slide 45
- Imaging brain metabolism. 2-deoxygluocose method radioactive
detection or positron emission tomography (PET) scanning, need
isotopes poor time resolution (Sokoloff 1977 J. of Neurochem.).
Functional magnetic resonance imaging (fMRI), second level time
resolution, signals related to changes in oxy/deoxyhemoglobin
potentially complicated (Ogawa et al. 1990 PNAS). Intrinsic signal
imaging more direct spectroscopy of brain signals related to
changes in oxy/deoxyhemoglobin, can be performed with a video
camera (Grinvald et al. 1986 Nature).
- Slide 46
- 10 m Synapses are on average 13 m from capillaries. RBC supply
rates are normally ~100 cells/sec. Acute reduction in supply rate
by >90% leads to damage within 10 min, which can reverse if
reperfusion occurs early. Zhang et al. 2005
- Slide 47
- Scale bar=10 umregion1 ctr at 49_54 10 m Control 10 min 30 min
1 hr 2 hr 3 hr Irreversible ischemia; red vessels, green dendrites
(Murphy lab). clot
- Slide 48
- 635 nm light 1) Reduced reflection, increased absorbance with
elevated deoxyhemoglobin in active areas. 2) General increase in
blood volume and oxyhemoglobin in surrounding areas leads to large
late positive global signal. Intrinsic optical signals, light
scattering provides a reflection of neuronal activity. Stim 1 sec
Reflected light 2) General blood volume. 1)Local deoxyhemo- globin
signal.
- Slide 49
- From Grinvald and Bonhoeffer OPTICAL IMAGING OF ELECTRICAL
ACTIVITY BASED ON INTRINSIC SIGNALS AND ON VOLTAGE SENSITIVE DYES
THE METHODOLOGY 2001 Sources of intrinsic optical signals.
- Slide 50
- Change in light scattering in response to forelimb
stimulation.
- Slide 51
- Neurotransmitters: small molecule and neuropeptide.
- Slide 52
- Small molecule Neurotransmitters (MW