Cholinergic and adrenergic neurotransmission

Dr. Erzsébet Tóth

7. October 2015.

Department of Medical Biochemistry Semmelweis University

Formation of acetylcholin in the nerve terminal in the cytoplasm

Choline acetyltransferase

Kolin-acetiltranszferáz

Cholinergic neuorotransmission

ph

osp

hatidyl ch

olin

e

sp

hin

go

mye

lin

ph

osp

hatidyl e

tha

no

am

ine

ph

osp

hatidyl se

rin

e

sources of choline:

absorption from food

plasma membrane degradation

uptake from synaptic gap

PC

Voltage-operated

High affinity choline - Na+ symporter

action

potential

Acetylcholinesterase

in synaptic cleft

- two important sites:

- one binding site for quaterner N

- at the active site carbonyl C is

attacked

- active site of enzyme contains:

Ser, His, Glu

- covalent ester bond intermediate

between Ser of enz. and acetate

- reaction mechanism of ACE is

similar to serine proteases

H2O

Acetylcholinesterase inhibitors

Gyógyszerek = ideggázok = nerve gases,

medicines rovarölők = insectisides

dohány

nicotine

Nicotiana tabacum

Ligand-operated

cation channel is the

pentamer nicotinic

receptor:

Na+ and Ca2+ channel

Each nicotinic receptor contains

5 subunits, it is pentamer.

α-subunit have10 isotypes

β-subunit have 5 isotypes

In neurons there are

5α homopentamer, or

2 α and 3 β heteropentamer

In skeletal muscle there is

(2α)βγδ heterotetramer

Ach always binds to α-subunit

The No of α-subunits equal the No

of bound Ach

One subunit has 4 transmembrane

segments, 4 α-helices.

Acetylcholine is an ancient signal transducing molecule:

found in bacteria, algae, protozoons, plants, animals, humans it has a regulatory role in: proliferation, migration, secretion, survival, apoptosis

Nicotinic receptor exist in the human cell, but the cell doesn’t produce acetylcholine: skeletal muscle: α1α1β1γδ neuromuscular junction macrophages, microglia: α7 pentamer (proliferation ↓, IL-6,12, TNF-α secretion) astrocyte: α7 pentamer (brain function) blood vessel smooth muscle: α 2-5,7,10 pentamers (proliferation)

Acetylcholine is synthetized, secreted, has receptor, has ACE: lymfocytes: T-lymph. proliferation, differentiation, selection, B-lymph. growth, Ab secretion ↓ keratinocytes: proliferation, apoptosis, differentiation, adhesion, motility blood vessel endothel: α3,5,7,10 + β2,4 pentamer: angiogenesis, smooth muscle proliferation respiratory tract: many kind of cells, receptors and effect CNS = central nervous system: transmitter liberation, exitability, sleeping, neural integration, awakeness, tiredness, pain, eating, cognitive functions α homopentamers and 2α3β heteropentamers

Effect of Ach in respiratory tract – not to study

Skeletal muscle contraction

1.) action potential →

Na+ channels and VOOC open →

Ach vesicles’ exocytosis

2.) Ach binds to N-type Ach receptor

3.) Na+ enters through nicotinic rec.

→ postsynaptic potential →

voltage operated Na+ channel opens →

action potential on sarcolemma →

T-tubules L-vocc and

SER ryanodin receptor (RR)

functional interaction allows

Ca2+ release from SER →

Ca2+ binding to troponin C →

conformational change in

troponin I, T, tropomyosin →

F-actin binding site becomes free

to interact with myosin → contraction

We do not ask it in biochemistry

Kerti susulyka

(Inocybe fastigata)

Every Inocybe species

is either poisonous or

not eatable

Inocybe lanuginella Clitocybe dealbata

Légyölő galóca

(Amanita muscaria)

Muscarine and muscarine

containing mushrooms

M3 receptor

Muscarinic receptors are heterotrimer G protein-coupled

7 transmembrane segment type receptors, 5 isotypes exist

INHIBITORY EFFECT OF ACETYLCHOLIN IN HEART – 1-2.

+ inotropic, + chronotropic, + dromotropic effect

hyperpolarization,

slower heart beat =

bradycardia =

lassabb szívverés

- chronotropic: pacemaker frequency↓

in sinoatrial node

- dromotropic: conduction velocity in

AV-node ↓

- inotropic : force of contraction ↓

especially in atria

• Contraction effect of acetylcholine in

gastrointestinal smooth muscle,

• secretion in digesting glands, • narrowing of pupil phincters (opening of

Schlemm channels and

decreasing of inner pressure in eye)

Acetylcholine activates secretion of

digesting juice in digesting glands during

signal transduction of vagus nerve (X brain

nerve), Ach bind to M3 (muscarinic 3)

receptors

Acetylcholine causes smooth muscle contraction in certain

muscles

MLC = myosine light chain

Smooth muscle contraction and relaxation

Ach

M3,5-rec

Gq

IP3

in SR

:adrenalin,

noradrenalin

GI=

gastrointestinal tract

longitudinal muscles,

pupilla sphincter

GI tract,

bronchi, genitourinal tract,

blood vessel smooth

muscle

AC

PKA

Figure 15-12a

Molecular Biology of the Cell

(© Garland Science 2008)

.

M3/M5 muscarinic rec. = GPCR = G-protein-coupled receptor

cGMP activates protein kinase G.

PKG phosphorylates and activates plasmamembrane Ca2+-ATPase and Na+/Ca2+ antiporter and SER

Ca2+ -ATP-ase, which removes Ca2+ from cytoplasm.

PKG phosphorylates Rho-dependent kinase inhibitory protein, it stops inhibition, myosin light chain

phosphatase can dephosphorylate MLC, so inactivates it, no contraction.

Ca2+ channel is inactivated, the ion does not enter.

PKG phosphorylates K+ channel, it opens, cell is hyperpolarized,

Smooth muscle is relaxed.

and Erabutoxin

(from sea snake)

are blockers

in skeletal muscle,

κ-bungarotoxin inhibits

in neurons

Toxins of some

animals and bacteria

effecting on

cholinergic signal

transduction

Botulismus:

recep

tor

localization signal

transduction

effect agonist antagonist

M1 Postsynaptic neuron:

striatum, cortex, hippocampus

Gq, PLC +

Ca2+

transmitter

release

atropin

M2 heart, bronchus

presyn. parasymp.neur.

hypothalamus, brain

Gi, AC –

Gi, K-channel.

- chronotr. bronchoconstr.

transm.liberation inhib,

hypothermia, anelgesia

atropin

M3 GI tract longitud. muscle,

bronchus

digestive glands, brain,

pupilla sphincter,

Gq, PLC +,

Ca2+

contraction

secretio, anelgesia

pupilla narrowing

neostigmin

physostigmin

pilocarpin

atropin

M4 striatum,hippocampus, cortex,

spinal cord pre- and postsyn.

Gi, AC –

Gi, K-channel.

DA liberation regulation

anelgesia…

atropin

M5 dopaminergic neurons, basal

ganglions, blood vessels

Gq, PLC +

Ca2+

DA liberation regul.

brain arthery relax.

atropin

Nm skeletal muscle Na+-channel contraction tubocurarin,

cobrotoxin,

α-bungarotoxin

Nn nervous system,

ganglion, adrenal gland

Na+/Ca2+ -

channel

action potential Κ-bungarotoxin

Nn macrophage

astrocyte,

smooth muscle.

Na+/Ca2+ -

channel

proliferation↓,

brain function

prolif.↑

Κ-bungarotoxin

Nn lymphocyte,

keratinocyte

endothel

respiratory tract

Na+/Ca2+ -

channel

prolif. diff. selection

prolif. diff. adhesion

angiogenesis

different effects

Κ-bungarotoxin

Catecholamines,

adrenergic and noradrenergic signal transduction

= catechol

Syntehsis of

catecholamines:

dopamine,

noradrenaline,

adrenaline

dopamine is a

neurotransmitter

in brain stem in

basal ganglions:

striatum,

substantia nigra

most important

sympathetic

neurotransmitter

Mainly hormon in

adrenal medulla,

but neurotransmitter

in brain, too

Noradrenerg idegvégződés

Noradrenergic nerve terminal

Na+

stress

↓

hypothalamus

↓

CRH

↓

ACTH

↓

adrenal cortex

↓

side chain cleavage enz. act.

induction of tyrosine hydroxylase

↓

cortisol synth. and secr.

↓

blood in adrenal gland

↓

adrenal medulla

↓

induction of phenyletahnolamine

N-methyltransferase

↓

synthesis of adrenaline ↑

slowest process

sympathetic

activation

Ach

↓

adrenal medulla

↓

action potential

↓

IC [Ca2+] ↑

↓

adrenaline

liberation

fast, but

short duration

of effect

sympathetic fibers

↓

action potential

↓

IC [Ca2+] ↑

↓

Ca2+-camodulin

dependent

protein kinase

is activated

↓

tyrosine hydroxylase act. ↑

↓

synthesis of chatecholamines

(noradrenaline) ↑

release of NA

↓ effects

in organs

fastest

Regulation of

synthesis of stress

hormons and

sympathetic

neurotransmitters

effects in other organs

effects in other

organs

MAO = monoamine oxidase

COMT = catechol

oxymethyltransferase

and either

aldehyde dehydrogenase

or aldehyde reductase

O2 + H2O

H2O2 + NH3

Degradation of

catecholamines

All adrenerg receptors are heterotrimeric G protein-coupled

7 transmembrane segment receptors

BETA2-adrenerg receptor:

β1,2,3 receptors activate,

α2 receptors inhibit

adenylyl cyclase

α1-receptor increases [Ca2+] intracellularly

α1D

Stimulatory effects of adrenalin/noradrenalin in cardiomyocytes, inhibitory effect of Ach

RyR = ryanodine receptor Ca2+ channel = Ca2+-csatorna

PLB = phospholambane = foszfolambán = Ca2+-ATP-áz reguláló fehérje/ regulating protein

AKAP = A kinase anchoring protein = PKA-horgonyzó fehérje

VOCC = voltage-operated Ca2+ channel = feszültségfüggő Ca2+-csatorna

VOCC

Adrenalin activates gluconeogenesis and glycogenolysis,

it inhibits glycolysis and glycogen synthesis in liver

Adrenalin activates gluconeogenesis and glycogenolysis,

it inhibits glycolysis and glycogen synthesis in liver

Control of lypolysis in white adipocytes

ATGL

AC and PKA are inhibited,

PDE is activated

FFA-albumin

liver

Alfa1-adrenerg receptor

α1-receptors are found in arteriols,

blood vessels contract

by the effect of adrenalin and noradrenalin

to increase blood pressure,

so α1-receptor antagonist drugs

can decrease the blood pressure.

1.) In different organs different ARs are dominated, mainly only these are mentioned:

α 1: arteriols, GI and GU sphincters contract

liver glycogenolysis

kidney Na+ reabsorption, skin sweating

adipocyte lipolysis and glycogenolysis

α2: GI longitudinal muscles relax, sphincters contract,

pancreas insulin secretion↑, glucagon secretion↓

β1: all the heart function increase, (β1: β2 = 80:20, also β3 and α1)

adipocyte lipolysis, glycogenolysis

kidney renin secretion from juxtaglomerular cells,

ghrelin secretion from stomach

β2 : bronchi, GI and GU smoothe muscles relax

liver: glycogenolysis, glyconeogenesis,

adipocyte lipolysis,

kidney renin secretion

β3: white adipocyte lipolysis ↑, brown adipocyte thermogenesis

2.) adrenerg receptor type in organs depends on species

e.g. human adipocytes β1 > β2 > β3 and also α1 and α2,

but rat β3 > β2 > β1 and also α1 and α2

3.) receptor types have different signal transduction and different effect

e.g. β 1,2,3 and α1 receptors activate, but α2 inhibit HSL

(hormonsensitive lipase) enzyme in adipocyte

4.) adrenerg receptor type depends on age, differentiation stage

e.g. in neonatal heart α1 receptors’ overproduction make growth,

heart hypertrophy

5.) altered metabolic state changes receptor types

e.g. in obesity α2/β1,2,3 ratio increases, consequently lipolysis decreases

e.g. in heart failure β1 receptor number ↓, while β2 and α1 receptors

non cAMP-dependent signal transduction↑

K+ channel opens,

Ca2+ channel closes

everything contracts

and moves

Different signal transduction routes of adrenergic receptors

• Speed of the synthesis e.g. testosteron, TNF-α → adipocyte β2-AR mRNA↑

• Desensitization happens due to repeated or continuous stimulus: a) AR-P (phosphorylated adrenergic receptor) → internalization → degradation translocation back e.g. β-AR → PKA → NA or A-bound AR-P → internalization α1-AR → PKC → NA or A- not bound AR-P → internalization b) AR mRNA and/or AR proteins are degraded

Quantity and sensitivity of adrenal receptors depends on:

Figure 15-51 Molecular Biology of the Cell

(© Garland Science 2008)

Localization and trafficking of

α2-adrenergic receptor subtypes

in cells and tissues (1999)

It depends on the target cell protein content, which proteins are phosphorylated, by this way the activity or amount of proteins are changed

PKA (cyclic AMP-dependent protein-kinase) substrates that are phosphorylated: adipocyte: HSL activated (perilipin also) TAG → 3 FA + glycerol → FFA ↑ heart muscle: L-VOCC opens→ Ca2+↑ → + inotropic effect phospholamban SER-ben → Ca2+-pump act. → during relaxation Ca2+ enters to SER ↑ → faster reuptake of Ca2+ → faster heart beat = tachycardia PFK-2/FBP → F2,6P2 ↑ → PFK-1 act. → glycolysis ↑ smooth muscle: MLCK → Ca2+ sensitivity↓ → relaxation liver: GS inact. → glycogen synthesis ↓ PFK-2/FBP → F2,6P2 ↓ → PFK-1 not act. and F1,6P2-ase not inact. → gluconeogenesis ↑ and glycolysis ↓ CREBP → in nucleus it induces: PC, PEPCK, F1,6P2-ase, G6P → gluconeogenesis ↑ PK inact. → glucose sparing PDHC inact. → from pyruvate AcCoA ↓ → glucose sparing ACC α inact. → malonyl-CoA ↓→ FA synthesis ↓ skeletal muscle: GPK act. → GP act. → glycogen degradation ↑ CREB → PGC -1α induction → mt. DNS replication ↑ → mt. number↑ → longer aerobic metabolism during excercise

The A-kinase anchoring proteins (AKAPs) are an important family of

scaffold molecules that are involved in the organization of multiprotein,

multivalent G-protein-signalling complexes. The example shown is that

of the AKAP gravin (also known as AKAP250), which associates

reversibly with the cell membrane in response to the activation of

protein kinase A (PKA) and the phosphorylation of gravin and the G-

protein-coupled receptor (GPCR; in this case, the 2-adrenergic

receptor) on its C-terminal cytoplasmic tail. The scaffold functions as a

'toolbox' of interacting molecules that affect the overall signalling of the

G-protein-based signalling complex. Molecules organized by gravin

include adenylyl cyclase (AC), the regulatory (R) and catalytic (C)

subunits of PKA, protein kinase C (PKC), Ca2+/calmodulin-dependent

protein kinase II (CaMKII), the GPCR, the receptor tyrosine kinase Src,

the phosphoprotein phosphatase PP2B, and the adaptor protein -

arrestin ( -arr). The G-protein subunits as well as the scaffold can bind

various cytoskeletal elements, such as microtubules and filamentous

actin networks. Other G-protein-interacting proteins include the

regulators of G-protein signalling (RGS), the activators of G-protein

signalling (AGS), and Partners of Inscuteable (Pins), which functions in

spindle formation. cAMP, cyclic AMP; P, phosphate.

Figure 15-36 Molecular Biology of the Cell (© Garland Science 2008)

Effect on gene

expression