Nerve cellsNeurotransmission across synapses

Biochemistry II

Lecture 6 2011 (E.T.)

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Nervous tissue

• 2,4% of adults body weight, of which 83% is the brain

• Large consumption of energy

• Consumption of 20% O2 from the total amount

• Content of specific complex lipids

• Rapid turnover of proteins• Types of cells:

CNS - neurons, astrocytes, oligodendrocytes, ependymalcells, microglia

PNS – neurons, Schwann cells, satelite cells

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capilary

neuron

astrocyte

microglia

oligodendrocyte

Types of the cells

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Endotel lining of vessels in brain

Glc

Kapilary beds in peripheral tissues Kapilary beds in CNS

Blood-brain barier

Free transport of substrates from blood is restricted

The all hydrophilic substrates must have transportion systemsOnly water and small lipophilic molecules are freely permeable

– free diffusion through intercellular space– pinocytosis (transcytosis)– glucose transporters

– numerous tight junctions limit free diffusion– lack of pinocytosis– the basement membrane is highly consistent– transporters GLUT have low efficiency

interstitial fluid

Continuousbasementmembrane

astrocytes

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Endotelial cells in brain

• Protect the brain

• Many drug-metabolizing enzyme systems

• Active P-glycoprotein pumps

• Specific transport systems for most of compounds

• Only small lipophilic molekules readily cross blood-brainbarrier by passive difusion

• Basement membrane surrounds cappilaries

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Glucose transporters in brain

• Endothelial membranes – GLUT 1

• Glial cells: GLUT1

• Neurons membranes – GLUT 3

• Rate of glucose transport is limited by KM for GLUT 1

Transport of glucose through capillary walls is much less efficient(blood-brain barrier).

When the glucose level drops below 3 mmol/l transport is not sufficient,hypoglycemic symptoms develop

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Transport of other molecules through blood –brain barrier

• Monocarboxylic acids (lactate, acetate, pyruvate, ketonebodies) – specific transporters

• Esential AA (phenylalanine, leucine, tyrosine, isoleucine,valine, tryptophan, methionine, histidine ) – rapidly enterCFS via a single amino acid transporters

• Non essential AA – transport is markedly restricted, theyare synthetized in the brain

• Vitamins – specific transporters

• Some proteins (e.g.*insulin, transferrin, IGF) cross theblood-brain barrier by receptor mediated endocytosis

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Lipids in the brain

• Fatty acids and other lipids (cholesterol, phospholipids,glycolipids ..) cannot penetrate blood-brain barrier

• All these lipids must be synthesized in the brain

• Only essential fatty acids (linolenic and linoleic) havespecific transporters

• Very long fatty acids are synthesized in brain (necessaryfor myeline formation)

• DHA is most abundant (22:6 (n-3))

• -oxidation, peroxisomal fatty acids oxidation occur inbrain

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Insulin receptors

• insulin receptors are observed in certain parts of themammalian brain (mostly on neurons)• brain is an insulinsensitive tissue and, in association with othernutrient and adiposity signals, such as fatty acids, amino acids,and leptin, probably plays an important role in the regulation ofenergy balance and glucose homeostasis.

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Energy production in brain

• metabolisms of glucose ( 100 g of glucose /d)

• utilisation of keton bodies (during starvation nearly 50% )

• Citric acid cycle

• Aerobic metabolism – consumption of 20% of O2

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Perikaryon – the metabolic centre ofneuron with intensive proteosynthesis, ishighly susceptible to low supply of oxygen.

Dendriteswith receptors of neurotransmitters.

Axon– the primary active transport of Na+ and K+ ions across

axolemma and voltage operated ion channels enablesinception and spreading of action potentials.

– axonal transport (both anterograde andretrograde) provides shifts of proteins, mitochondria,and synaptic vesicles between perikaryon andsynaptic terminals.

- myelin sheat enables the rapid saltatory conduction ofnerve impulses.

Axon terminals - synapses– neurotransmitters are released from synaptic vesicles

into the synaptic cleft by exocytosis.

Neurons

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Kinesin drifts proteins, synaptic vesicles, and *mitochondria inanterograde transport, dynein in retrograde transport.

Axonal transport

In the axon, there is a fast axonal transport along microtubules. It workson the principle of a molecular motor, via the motile proteins.

retrograde transport

anterograde transport

*Mitochondria are mobile organelles and cells regulate mitochondrial movement in order tomeet the changing energy needs of each cellular region. Ca2+ signaling, which halts bothanterograde and retrograde mitochondrial motion, serves as one regulatory input.

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Myelin sheaths are formed by wrappingof protruding parts of glial cells roundthe axons;oligodendrocytes (CNS)Schwann cells (PNS).Numerous plasma membranes aretightly packed so that the originalintracellular and extracellular spacescannot be differentiated easily.

Myelin membranes contain about 80 % lipids

(30% of cholesterol, 16% of cerebrosides).

The main structural proteins are

- proteolipidic protein,

- the basic protein of myelin (encephalitogen),

- high molecular-weight proteincalled Wolfram's protein.

Myelin

the "outer“ sides

cytoplasmic sides

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Myeline damage

• Demyelination disease - loss or damage of myelin, bothin the CNS and in the PNS

• E.g. Multiple sclerosis

The cause has yet to be determined; autoimmune procesdemyelination of white matter of brain tissue andmultifocal inflammation damaged nerve conductionalong the demyelinated area

Presence of oligoclonal immunoglobulins in cerebrospinalfluid

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Astrocytes

Roles of astrocytes:

• connected by gap junctions - syncitium

• support for migration of neurons, matrix that keeps them inplace

• synthesis and degradation of glycogen

• uptake and metabolism of neurotransmiters (e.g. GABA,glutamate)

• by phagocytoses take up debris left behind by cells

• control the brain extracellular ionic enviroment (Na+/K+-pump, K+-„uptake“, Na+-HCO3

– cotransport, transport ofgases.)

• secretion of neurotransmiters (NO, VIP /vasoactiveintestinal peptide/ PGE2)

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Metabolism of ammonia in brain

• Glutamate released by neurons into the synaptic cleft is takenup by astrocytes and recycled into the form of glutamine

• NH3 crosses the H-E barrier

• If the concentration of ammonia in blood is high, it isincorporated into Glu a Gln in astrocytes (glutamatedehydrogenase, glutamin synthetase).

• depletion of ATP, due to depletion of 2-oxoglutarate forCAC accumulation of Gln and Glu in astrocytes.

• Accumulation of Glu in astrocytes lieds to fall in transport ofGlu from synaptic cleft (and damage of neurons by excitotoxiceffects of Glu).

• Surplus of Gln results in damage of osmotic regulation inastrocyte, increased intake of water oedem of brain

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glutamate

Astroglial cell Neuron

Capilary

Blood-brainbarier

NH4+ NH3 NH3 NH4

+

glutamine glutamate

GABAglutamate

+ 2-oxoglutarateNH4+ NH3 NH4

+ NH3

ATP

ATP

Metabolism of ammonia in brain

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Nerve impulse

•Neurons are irritable cells

•The lipidic bilayer is practically impermeable to theunevenly distributed Na+ and K+ ions.

•The resting membrane potential is –70 mV on the innerside of the plasma membrane.

•This potential is maintained by Na+,K+–ATPase.

ligand-gated Na+/K+ channel,– voltage-operated Na+ channel, and– voltage-operated K+ channel.

Channels present in the membrane:

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-70

mV Spikepotential

repolarization

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Changes of membranepotential during anaction potential(propagation of nerveimpuls along themebrane)

Time(ms)

•stimulus triggers the depolarization

• if the depolarization reaches the „treshold value“ voltage operating Na+ channels will open,Na+ enters massively the cell, depolarization takes place

• this triggers the slow opening K+ channels, Na+ channels will slowly close, potassium fluxesinside the cell, repolarization takes place

• Na+/K+ pump restores the concentration gradients disrupted by action potential

•Action potential is propagated to the axon terminals

Na+ channelsopened

Treshholdpotential

Restingpotential

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Transmiting the nerve impuls across the synapticcleft

•Action potential travels down the axon to the axon terminal

•Each axon terminal is swollen, forming a synaptic knob

•The synaptic knob is filled with membrane –bounded vesiclescontaining a neurotransmitter

•Arrival of an action potential at the synaptic knob opens voltage-gated Ca2+-channels in the plasma membrane

•The influx of Ca2+ triggers the exocytosis of some of the vesicles

•The nerotransmitter is released into the synaptic cleft

•The neurotrabnsmitter bind to receptors on the postsynapticmembrane

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Synaptic transmission

Neurotransmitters act as chemical signals between nerve cellsor between nerve cells and the target cells.

The response to the neurotransmitter depends on the receptor type:– ionotropic receptors (ion channels) evoke a change in the

membrane potential - an electrical signal,– metabotropic receptors are coupled to second messenger

pathway, the evoked signal is a chemical one.

synaptic cleft

postsynapticmembrane

receptor

synaptic vesicles(synaptosomes)

voltage-gated Ca2+ channel

depolarization wave

Ca2+

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NeurotransmittersA large number (much more than 30) of neurotransmitters have been described.Many of them are derived from simple compounds, such as amino acids andbiogenic amines, but some peptides are also known to be importantneurotransmitters. The principal transporters:

Peripheral neurons

– efferentexcitatory acetylcholine

noradrenaline

– afferent sensory neuronsglutamateneuropeptides

Central nervous system

inhibitory GABA (at least 50 %)glycine (spinal cord)

excitatory glutamate (more than 10 %)acetylcholine (about10 %)dopamine(about 1 %, in the striatum 15 %)

serotoninhistamineaspartatenoradrenaline (less than 1 %,

but in the hypothalamus 5 %)adenosine

neuromodulatory endorphins, enkephalins,endozepines, delta-sleep inducing peptide,and possibly endopsychosins.

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Ionotropic receptors – ligand-gated ion channels (ROC), e.g.

excitatory – acetylcholine nicotinic - Na+/K+ channel,

– glutamate (CNS, some afferent sensory neurons)- Na+/Ca2+/K+ channel,

inhibitory – GABAA receptor (brain) - Cl– channel

Metabotropic receptors activating G proteins, e.g.

Gs protein – -adrenergic, GABAB receptor, dopamine D1,

Gi protein – 2-adrenergic, dopamine D3,acetylcholine muscarinic M2 (opens also K+ channel),

Gq protein – acetylcholine muscarinic M1,1-adrenergic.

Contrary to various types of hormone receptors, only two basal typesof neurotransmitter receptors occur:

Neurotransmitter receptors

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nicotinic muscarinic

Acetylcholine receptors

M1-M5

Metabotropic, act throuhg G-proteins

M1 –Gq protein, vegetative ganglia, CNS,b.exocrine glands

M2 –Gi, in heart, opens K+-chanels

M3 - Gq protein, in smooth muscle

M4 –Gi, v CNS

M5 –Gq, v CNS

Atropin is an acetylcholine antagonist atmuscarinic receptors

acetylcholine-operatedNa+/K+ channels ;

in the peripheral nervoussystem

in the dendrites of nearlyall peripheral efferentneurons (includingadrenergic neurons)

at neuromuscularjunctions ion the cytoplasmicmembrane of skeletalmuscles.

.

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Cholinergic synapse

ACETYLCHOLINE

membrane-boundacetylcholinesterase

choline

acetyl-CoAATP

depolarization wave

Ca2+

Na+

choline acetyltransferase(by axonal transport)

acetylcholine receptors

acetate

Increase in intracellular [Ca2+] activates Ca2+-calmodulin-dependent protein- kinase thatphosphorylates synapsin-1; its interaction with the membrane of synaptic vesiclesinitiates their fusion with the presynaptic membrane and neurotransmitter exocytosis.The membranes of vesicles are recycled.

reuptake

At neuromuscular junctions, the arrival of a nerve impulse releases about 300 vesicles (approx. 40000 acetylcholine molecules in each), which raises the acetylcholine concentration in the cleft morethan 10 000 times.

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Myastenia gravis

• Myastenia gravis is an aquired autoimmune disease causedby production of an antibody directed against theacetylcholine receptor in skeletal muscle

• The antibodies bind to the receptor forming receptor-antibody complex

• Complex is endocytosed and incorporated into lysosomes

Number of receptors is reduced

Muscle weakness

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Acetylcholine esterase

• Hydrolysis of acetylcholine to acetate and choline

• Serine hydrolase

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Inhibitors of acetylcholinesterase

• a) reverzible: carbamates (physostigmine,rivastigmine, neostigmine)

• b) irreverzible: organophosphates(diisopropylfluorphosphate, soman, sarin)

Binding of toxic organo phosphates occurs in two phases:

reversible ( it may be affected by reactivators)

ireversible – formation of covalent bond between organophosphate and an enzyme

N N

OO

NH

CH3

CH3

CH3

CH3

H

phyzostigmine

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Reversible phase

Irreversible phase

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N+

N+

O

N N

OH OH

Cl-

Cl-

Reactivators of acetylcholinesterase

N+

N

CH3

OHCl

-

pralidoxime

obidoxime

Oximes with pyridine heterocycle

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Function of reactivators

Regeneration of acetylcholin esterase

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•asymmetric pentamer of four kinds of membrane-spanning homologous subunits•activated by binding of two molecules of acetylcholine.

2-subunits bind twoacetylcholine molecules

the closed state

synapticcleft

– –

Na+ K+

2 2

changes in conformation, the channelundergoes frequent transitions between

open and closed states in few milliseconds

cytoplasm

a large inwardflow of Na+

a smaller outwardflow of K+

binding sites for local anaesthetics,psychotropic phenothiazines. etc.

D-Tubocurarine is an antagonist of acetylcholine that prevents channel opening.Succinylcholine is a myorelaxant that produces muscular end plate depolarization.

Acetylcholine nicotinic receptor – Na+/K+ channel

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Acetylcholine (cholinergic) receptorsof the peripheral efferent neurons

motor neurons parasympathetic sympathetic(neuromuscular junction) system system

Most postganglionicneurons of the sympatheticpath are adrenergic

Adrenergicreceptors

N

NN

N

N

N

M1

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Adrenergic synapseNeurotransmitter of most postganglionic sympathetic neuronsis noradrenaline.

depolarization wave

Ca2+

adrenergic receptors inmembranes of the target cells

DA -hydroxylasesynaptic vesicles

(axonal transport)

NORADRENALINE

presynapticadrenergic

receptorsmitochondrial

monoamine oxidase

partial reuptake

Varicosities of the postganglionic sympatheticaxons are analogous to the nerve terminals.

extracellular COMT(catechol O-methyltransferase)

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Synthesis and storage of catecholamines

• Synthesis from tyrosine DOPA dopamin in cytosol

• Dopamin is transported into vesicles (ATP dependenttransport)

• Vesicles are transported to nerve terminals

• -hydroxylation to noradrenalin occurs in vesicles

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of all types are receptors cooperating with G proteins.

-Adrenergic receptorsAfter binding an agonist, all types of -receptors activate Gs proteins so

that adenylate cyclase is stimulated, cAMP concentration increases,and proteinkinase A is activated. Particular types differ namely intheir location and affinity to various catecholamines:1 are present in the membranes of cardiomyocytes,2 in the smooth muscles and blood vessels of the bronchial stem,3 in the adipose tissue.

2-Adrenergic receptorsThe effect is quite opposite to that of -receptors, binding of

catecholamines results in the interaction with Gi protein,decrease in adenylate cyclase activity and in cAMP concentration.

1-Adrenergic receptorsactivate Gq proteins and initiate the phosphatidylinositol cascade by

stimulation of phospholipase C resulting in an increase ofintracellular Ca2+ concentration and activation of proteinkinase C.

Adrenergic receptors

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Adrenergic receptors 1, 2, and 3

AMP cyclase receptor

Gasbg

noradrenaline/adrenaline

ATP

AMP

cAMP

inactiveproteinkinase A

active proteinkinase A

phosphodiesterases

phosphorylations

The typical effects of -stimulation:1 – tachycardia, inotropic effect in the myocard,2 – bronchodilation, vasodilation in the bronchial tree,,3 – mobilization of fat stores, thermogenesis.

H2O

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Adrenergic receptors 2 a 1

Receptors 2 Receptors 1

cAMP decrease

IP3 and diacylglycerol

Gi protein Gq protein

adenylate cyclase phospholipase C

PL C

increase in [Ca2+]activation of PK C

The typical effects of adrenergic

2-stimulation: 1-stimulation:glandular secretion inhibited vasoconstriction

bronchoconstrictionmotility of GIT inhibited

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Cl–

––

––

–– –

12

2

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Inhibitory GABAA receptorligand-gated channel (ROC) for chloride anions. The interaction with-aminobutyric acid (GABA) opens the channel. The influx of Cl– isthe cause of hyperpolarization of the postsynaptic membrane and thus

its depolarization (formation of an action potential) disabled.

The receptor is a heteropentamer.Besides the binding site for GABA, it has at least eleven allosteric modulatorysites for compounds that enhance the response to endogenous GABA – reductionof anxiety and muscular relaxation: anaesthetics, ethanol, and many useful drugs,e.g. benzodiazepines, meprobamate, barbiturates. Some ligands compete for thediazepam site or act as antagonists (inverse agonists) so that they cause discomfortand anxiety, e.g. endogenous peptides called endozepines.

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GABA (-aminobutyric acid) is the major inhibitory neurotransmitter in CNS.Gabaergic synapses represent about 60 % of all synapses within the brain.

GABA

mitochondrial synthesisof GABA from glutamate

depolarization wave

Ca2+

GABA / benzodiazepinereceptors

uptake of GABA into glial cellsand breakdown to succinate

partial reuptake(transporters GAT 1,2,3,4)

Inhibitory synapse

In the spinal cord and the brain stem, glycine has the similar function as GABA in thebrain. The inhibitory actions of glycine are potently blocked by the alkaloid strychnine, a

convulsant poison in man and animals.

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Receptors for the major neurotransmitters

Receptors cooperating with G-proteinsIon channels(ROC) Gs (cAMP increase) Gi (cAMP decrease) Gq (IP3/DG formation)

Na+/K+ – acetylcholinenicotinic

acetylcholinemuscarinic M2,4

adrenergic β1, β2, β3 adrenergic α2 adrenergic α1

Na+/Ca2+/K+

– glutamate ionophorsglutamate mGluRgroup II and III

glutamate mGluRgroup I

dopamine D1,5 dopamin D3,4 dopamine D2

– serotonin 5-HT3 serotonin 5-HT4,6 serotonin 5-HT1 serotonin 5-HT2

– histamine H2 histamine H3,4 histamine H1

tachykinin NK-1for substance P

Cl– – GABAA

– glycineGABAB (metabotropic)

acetylcholinemuscarinic M1,3,5

–

– –

–

–

–

–

–