SIGNAL TRANSDUCTION BY ADRENERGIC AND CHOLINERGIC RECEPTORS

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SIGNAL TRANSDUCTION BY ADRENERGIC AND CHOLINERGIC
RECEPTORS

• Andy Catling Ph.D.
• Department of Pharmacology Room 5238
• acatli_at_lsuhsc.edu
• 568-4740

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Adrenergic Receptors

• Alpha Receptors
• ?1 Contraction of vascular and genitourinary
  smooth muscle.
• ?2 Contraction of vascular smooth muscle
  decreased insulin secretion
• aggregation of platelets pre-synaptic
  inhibition of NE.
• Beta Receptors
• ?1 Positive inotropic and chronotropic effects
  on the heart.
• ?2 Relaxes vascular, bronchial, gastrointestinal
  and genitourinary
• smooth muscle stimulates glyconenolysis
  and gluconeogenesis in
• the liver.
• ?3 Lipolysis in adipose tissue.

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Cholinergic Receptors

• Nicotinic Receptors
• NM (muscle) Depolarization of NMJ Skeletal
  muscle contraction.
• NN (neuronal) Activation of post ganglionic
  neurons.
• Muscarinic Receptors
• M1 Depolarization of autonomic and CNS neurons
• M2 Negative inotropic and chronotropic effects
  on
• the heart.
• M3 Stimulates sweat, bronchial, salivary and
  gastric acid
• secretions Increased NO production from
  vascular
• endothelium and vasorelaxation.

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How does this work?

• Different receptors can have reinforcing or
  opposing functions
• e.g. ?1 adrenergic receptors stimulate
  contraction of vascular smooth muscle whereas b2
  adrenergic and muscarinic M3 receptors both cause
  relaxation.
• e.g. ?1 adrenergic receptors stimulate
  contraction of heart muscle, whereas muscarinic
  M2 receptors inhibit myocardial contraction.
• Goal of these lectures begin to understand the
  signal transduction mechanisms by which
  specificity is established. Note that this is
  still a work-in-progress the human body is
  complex (!) and there are both gaps in our
  knowledge and exceptions to general rules

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Components of Signal Transduction

• Signal transduction within cells is accomplished
  by
• combinations of
• 1st Messenger (extracellular signals e.g.
  epinephrine, acetylcholine)
• Receptor
• Effectors (e.g. adenylyl cyclase, phospholipases,
  kinases, ion channels etc)
• 2nd messengers (cAMP, cGMP, inositol
  triphosphate, diacylgycerol, Ca2 etc)
• Downstream effectors required for specific
  functional outputs (e.g. muscle contraction,
  secretion)

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How does this work?

• Specificity results from
• Differential expression and localization
  (junctional vs extra junctional) of receptors
• Different receptors couple to different signal
  transducers
• Signal transducers/2nd messengers couple to
  different effectors in different tissues
• Integration of reinforcing and antagonistic
  signals

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b Adrenergic Receptors

• b receptors differ in their location and
  sensitivity to Epinephrine and Norepinephrine
  (simplified!)
• b1 Myocardium ENE
• b2 Smooth muscle E (essentially no affinity
  for NE)
• b3 Adipose tissue NEgtE
• i.e. tissue response to agonist is governed by
  expression of receptor subtypes and ligand
  present
• All three b adrenergic receptors function through
  a major class of signal transducer G-proteins
• G-proteins couple b adrenergic receptors to
  adenylyl cyclase
• b agonists increase intracellular cyclic AMP
  levels and protein kinase A activity, which in
  turn regulate downstream effectors

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G-protein Activation-Deactivation Cycle
??GDP ??
??GDP ??
GTP
Pi
GDP
GPCR
??GTP ??
Effectors
Effectors
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Adrenergic Receptors

• Beta Receptors
• ? 1 Receptors
• Gs (stimulatory) Activation of adenylyl cyclase
  and increased cAMP levels.
• Positive inotropic and chronotropic effects on
  the heart speeds conduction across the AV node.
• Agonist Dobutamine
• Antagonist Atenolol

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b1 adrenergic receptors function through Gs to
stimulate the effector adenylyl cyclase to
produce the 2nd messenger cyclic AMP

• Activated Gs
• stimulates adenylyl cyclase to produce
• cAMP
• enhances activation of voltage gated Ca2
• channels in the plasma membrane
• cAMP
• activates protein kinase A, which directly
• phosphorylates proteins (e.g. troponin I)
• essential for cardiac muscle contraction
• stimulates sodium/potassium influx which
• opens voltage-gated Ca2 channels
• inhibits uptake of Ca2 into cellular stores
• cAMP hydrolyzed by phosphodiesterases

Overall effect increased intracellular Ca2
concentration and phosphorylation of contractile
proteins. Result cardiac muscle cells
expressing b1 receptors contract in response to
epinephrine or norepinephrine.
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Adrenergic Receptors

• Beta Receptors
• ??2 Receptors
• Gs Activation of adenylyl cyclase and increased
  cAMP levels.
• Relaxes vascular, bronchial, gastrointestinal and
  genitourinary smooth muscle, stimulates the
  uptake of potassium into skeletal muscle,
  stimulates glycogenolysis and gluconeogenesis in
  the liver.
• Agonist Terbutaline
• Antagonist Propranolol

Why does b1 stimulation cause contraction in
cardiac muscle while b2 stimulation causes
relaxation of smooth muscle both elevate cAMP?
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Different downstream effectors different
responses
EPI, b1, cardiac muscle
EPI, b2, smooth muscle
protein kinase A
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Different downstream effectors different
responses
SMOOTH MUSCLE
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Adrenergic Receptors

• Beta Receptors
• ? 3 Receptors
• Activate Gs protein, stimulates adenylate cyclase
  and increases cAMP levels. cAMP activates PKA
  which stimulates the lipase activity i.e. another
  context-specific effector
• Adipose tissue Lipolysis.

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Adrenergic Receptors

• Beta Receptors summary
• ? Receptors
• b1, b2 and b3 ALL activate Gs which stimulates
  adenylyl cyclase and increases cAMP levels.
• cAMP activates protein kinase A
• Outcome depends on what PKA phosphorylates e.g.
  Troponin in cardiac muscle (contraction) MLCK in
  smooth muscle (relaxation) lipase in adipose
  tissue

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Adrenergic Receptors

• Alpha Receptors
• ?1 Contraction of vascular and genitourinary
  smooth muscle.
• ?2 Contraction of vascular smooth muscle but
  also indirect
• effects that lead to vasodilation. Also
  decreased insulin
• secretion, aggregation of platelets.

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Adrenergic Receptors

• a1 and b2 receptors both signal through
  G-proteins, yet can cause opposite effects on the
  same tissue (e.g. genitourinary smooth muscle).
• How?

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a1 and b2 signal through different G-proteins
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Adrenergic Receptors

• ?1 Receptors
• a1 receptors coupled to Gq not Gs
• Gq activates phospholipase C (PLC) causing
  production of inositol triphosphate (IP3) and
  diacyglycerol (DAG) from inositol phospholipids
• Gq-linked receptor operated calcium channel
• Overall effect is to increase intracellular
  calcium
• Calcium-calmodulin stimulates myosin light chain
  kinase activity and hence contraction of vascular
  and genitourinary smooth muscle
• Agonist Phenylephrine
• Antagonist Prazosin

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Epi, NE
Interstitial fluid
DAG
PLC
Gq
? 1
GTP
?
GDP
Contraction of vascular and genitourinary smooth
muscle
PIP2
?
IP3
?
Ca2
IP3
IP3R

Intracellular calcium pools
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Different downstream 2nd messengers and
effectors different responses
e.g. vascular or genitourinary smooth muscle
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IMPORTANT..direction of response depends upon
ligand concentration
e.g. in vascular smooth muscle
b2
a1
Gs, cAMP, VASODILATION
a1
b2
LOW EPI
b2
a1
Gq, Ca2, overcomes cAMP effects,
VASOCONSTRICTION
HIGH EPI
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Adrenergic Receptors

• a2 adrenergic receptors on vascular smooth
  muscle cause contraction
• How?

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Adrenergic Receptors

• direct effect on vascular smooth muscle is
  contraction mediated by extra-junctional ?2
  receptors
• NE or Epi stimulation of a2 engages Gi/o classes
  of G-protein
• Gi/o inhibits adenylyl cyclase thus decreasing
  cAMP levels
• Gi/o increases Ca2 influx
• Decrease in cAMP allows calcium-calmodulin
  stimulation of MLCK activity, causing contraction

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Gi and Gs have opposite effects on adenylyl
cyclase activity
Epi, a2 adrenergic receptor
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Direct effect of a2 on vascular smooth muscle
contraction
Vascular smooth muscle
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Adrenergic Receptors

• BUT a2 adrenergic receptors also can cause
  vasodilationon
• How?

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Indirect effect of a2 on vascular smooth muscle
relaxation and vasodilation
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Adrenergic Receptors

• Pre-synaptic ?2 receptors indirectly cause
  vasodilation
• stimulation of pre-synaptic a2 receptors by NE or
  EPI inhibits release of NE at the synapse
• NE concentration in the adrenergic synapse is
  reduced decreasing stimulation of post-synaptic
  a1 receptors
• Less post-synaptic a1/Gq activation, translates
  into less calcium-calmodulin stimulation of MLCK
• Relaxation

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Cholinergic Receptors

• Muscarinic Receptors
• M1 Depolarization of autonomic and CNS neurons
• M2 Negative inotropic and chronotropic effect
  on the heart.
• M3 Smooth muscle contraction with ONE
  EXCEPTION cause
• vascular smooth muscle relaxation and
  vasodilation
• Glandular secretion
• Also M4 and M5.
• Nicotinic Receptors
• NM (muscle) Depolarization of NMJ Skeletal
  muscle contraction.
• NN (neuronal) Activation of post ganglionic
  neurons.

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Multiple acetylcholine-mediated effects how?

• Different subtypes of cholinergic receptors in
  different tissues.

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Cholinergic Receptors

• Muscarinic Receptors
• M1 Autonomic ganglia, CNS, some secretory
  glands. Cause
• depolarization of autonomic and CNS neurons
• M2 Heart, CNS. Cause negative inotropic and
  chronotropic effects
• on the heart
• M3 Smooth muscle vascular endothelium and
  secretory glands.
• Cause smooth muscle contraction glandular
  secretion BUT
• also vasodilation
• i.e. as for adrenergic responses, tissue response
  is governed by
• expression of specific receptor subtypes

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What accounts for the differences in
Acetylcholine-mediated effects?

• Different subtypes of cholinergic receptors in
  different tissues.
• Different receptors are coupled to different
  G-proteins and hence different effectors.

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Different muscarinic receptors couple to
different G-proteins

• Muscarinic Receptors all G-protein linked
• M1 Gq/11 Gastric secretion in parietal cells
  and depolarization
• of autonomic and CNS neurons
• M2 Gi Negative inotropic and chronotropic
  effect on the heart.
• M3 Gq/11 Stimulates smooth muscle contraction
  sweat, bronchial
• and salivary secretions paradoxical
  vasodilation.

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Cholinergic Receptors

• Muscarinic Receptors
• M1 Receptors
• Gq/11 Activation of phospholipase C generates
  DAG and
• IP3 IP3 increases intracellular
  calcium
• i.e. M1 and a1 have similar signaling mechanism

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Acetylcholine
Interstitial fluid
DAG
PLC
Gq
M1
GTP
?
PKC
GDP
Neurons Depolarization of autonomic and CNS
neurons
PIP2
?
IP3
?
Ca2
IP3
IP3R

Intracellular calcium pools
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Cholinergic Receptors

• Muscarinic Receptors
• M2 Receptors
• Gi inhibition of adenylyl cyclase and decreased
  cAMP
• M2 and a2 have similar signaling mechanism
• Reduced PKA phosphorylation of troponin I,
  negative inotropic and chronotropic effect on the
  heart (i.e. antagonistic to b1)

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Cardiac Muscle
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Cholinergic Receptors

• Muscarinic Receptors
• M3 Receptors
• Gq/11 Activation of PLC, hydrolysis of IP3 and
  increased intracellular calcium, similar to M1.
• Secretion (bronchial, sweat and salivary glands,
  gastric acid) contraction of most smooth muscle
• Paradoxical relaxation of vascular smooth muscle
  and vasodilation result of increased synthesis
  of NO and PGI2 in vascular endothelium

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Indirect effect of M3 stimulation on vascular
smooth muscle vasodilation
M3
Gq/11
Guanylyl cyclase
COX cyclo-oxygenase PCS prostacyclin synthase
Gs
Vasodilation
Simplistically the endothelial cell converts a
Gq response (increased Ca2) to a Gs response
(increased cyclic AMP)
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Cholinergic Receptors

• Nicotinic Receptors Cation Channels
• NM Depolarization of NMJ Skeletal muscle
  contraction.
• NN Activation of post ganglionic neurons.

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Cholinergic Receptors

• Nicotinic Receptors Cation Channel
• NN Type
• Autonomic ganglia Activation of post ganglionic
  neurons in autonomic ganglia.
• Agonist Nicotine
• Antagonist Trimethaphan

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K Na
Ach
Ach
Ach
Ach
Binding
K Na
Open
NN nicotinic receptors Heteropentamers of ? and
? subunits or Homopentamers of ? subunits.
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Cholinergic Receptors

• Nicotinic Receptors Cation channel
• NM Type
• Neuromuscular Junction Depolarization of NMJ
  Skeletal muscle contraction.
• Heteropentamers of a, b, g and d subunits
• Agonist Acetylcholine
• Antagonist Tubocurarine

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Nicotinic Receptor (NM)
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