SIGNAL TRANSDUCTION BY ADRENERGIC AND CHOLINERGIC RECEPTORS Andy Catling Ph.D. Department of Pharmacology Room 5238

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.

Cholinergic Receptors Nicotinic Receptors: N M (muscle): Depolarization of NMJ; Skeletal muscle contraction. N N (neuronal): Activation of post ganglionic neurons. Muscarinic Receptors: M 1 : Depolarization of autonomic and CNS neurons M 2 : Negative inotropic and chronotropic effects on the heart. M 3 : Stimulates sweat, bronchial, salivary and gastric acid secretions; Increased NO production from vascular endothelium and vasorelaxation.

How does this work? Different receptors can have reinforcing or opposing functions: e.g.  1 adrenergic receptors stimulate contraction of vascular smooth muscle whereas  2 adrenergic and muscarinic M 3 receptors both cause relaxation. e.g.  1 adrenergic receptors stimulate contraction of heart muscle, whereas muscarinic M 2 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

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

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/2 nd messengers couple to different effectors in different tissues Integration of reinforcing and antagonistic signals

 Adrenergic Receptors  receptors differ in their location and sensitivity to Epinephrine and Norepinephrine (simplified!):  1MyocardiumE=NE  2Smooth muscleE (essentially no affinity for NE)  3Adipose tissueNE>E i.e. tissue response to agonist is governed by expression of receptor subtypes and ligand present All three  adrenergic receptors function through a major class of signal transducer: G-proteins G-proteins couple  adrenergic receptors to adenylyl cyclase:  agonists increase intracellular cyclic AMP levels and protein kinase A activity, which in turn regulate downstream effectors

 GDP +   GDP   GTP +  GTP GDP PiPi GPCR* Effectors G-protein Activation-Deactivation Cycle Effectors

Adrenergic Receptors Beta Receptors  1 Receptors G s (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

 1 adrenergic receptors function through G s to stimulate the effector adenylyl cyclase to produce the 2 nd messenger cyclic AMP Activated G s : - stimulates adenylyl cyclase to produce cAMP - enhances activation of voltage gated Ca 2+ 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 Ca 2+ channels - inhibits uptake of Ca 2+ into cellular stores - cAMP hydrolyzed by phosphodiesterases Overall effect: increased intracellular Ca 2+ concentration and phosphorylation of contractile proteins. Result: cardiac muscle cells expressing  1 receptors contract in response to epinephrine or norepinephrine.

Adrenergic Receptors Beta Receptors  2 Receptors G s : 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  1 stimulation cause contraction in cardiac muscle while  2 stimulation causes relaxation of smooth muscle – both elevate cAMP?

protein kinase A EPI,  1, cardiac muscle Phosphorylation of contractile machinery proteins: e.g. Troponin I CONTRACTION EPI, , smooth muscle Troponin I absent from smooth muscle. PKA phosphorylation of a different target, myosin light chain kinase, inhibits myosin function. RELAXATION Different downstream effectors: different responses

SMOOTH MUSCLE CONTRACTION RELAXATION EPI,  2, G s

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.

Adrenergic Receptors Beta Receptors: summary  Receptors  1,  2 and  3 ALL activate G s 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

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.

Adrenergic Receptors  1 and  2 receptors both signal through G-proteins, yet can cause opposite effects on the same tissue (e.g. genitourinary smooth muscle). How?

 1 and  2 signal through different G-proteins

Adrenergic Receptors  1 Receptors:  1 receptors coupled to G q not G s G q 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

PLC Gq  1 1 Interstitial fluid PIP 2 DAG IP 3 IP 3 R Ca 2+ GTP GDP Intracellular calcium pools    Epi, NE Contraction of vascular and genitourinary smooth muscle

Different downstream 2 nd messengers and effectors: different responses e.g. vascular or genitourinary smooth muscle RELAXATION EPI, , G s EPI, , G q CONTRACTION

IMPORTANT…..direction of response depends upon ligand concentration e.g. in vascular smooth muscle LOW EPI G s, cAMP, VASODILATION     G q, Ca2+, overcomes cAMP effects, VASOCONSTRICTION HIGH EPI

Adrenergic Receptors  adrenergic receptors on vascular smooth muscle cause contraction How?

Adrenergic Receptors direct effect on vascular smooth muscle is contraction mediated by extra-junctional  2 receptors: NE or Epi stimulation of  2 engages G i/o classes of G-protein G i/o inhibits adenylyl cyclase thus decreasing cAMP levels G i/o increases Ca 2+ influx Decrease in cAMP allows calcium-calmodulin stimulation of MLCK activity, causing contraction

G i and G s have opposite effects on adenylyl cyclase activity Epi,  2 adrenergic receptor

Direct effect of  2 on vascular smooth muscle: contraction Vascular smooth muscle CONTRACTION Epi,  GiGi

Adrenergic Receptors BUT  adrenergic receptors also can cause vasodilationon How?

Presynaptic Receptors (Autoreceptors) Indirect effect of  2 on vascular smooth muscle: relaxation and vasodilation

Adrenergic Receptors Pre-synaptic  2 receptors: indirectly cause vasodilation stimulation of pre-synaptic  2 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  1 receptors Less post-synaptic  1/Gq activation, translates into less calcium-calmodulin stimulation of MLCK Relaxation

Cholinergic Receptors Muscarinic Receptors: M 1 : Depolarization of autonomic and CNS neurons M 2 : Negative inotropic and chronotropic effect on the heart. M 3 : Smooth muscle contraction with ONE EXCEPTION: cause vascular smooth muscle relaxation and vasodilation; Glandular secretion Also M 4 and M 5. Nicotinic Receptors: N M (muscle): Depolarization of NMJ; Skeletal muscle contraction. N N (neuronal): Activation of post ganglionic neurons.

Multiple acetylcholine-mediated effects: how? Different subtypes of cholinergic receptors in different tissues.

Cholinergic Receptors Muscarinic Receptors: M 1 : Autonomic ganglia, CNS, some secretory glands. Cause depolarization of autonomic and CNS neurons M 2 : Heart, CNS. Cause negative inotropic and chronotropic effects on the heart M 3 : 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

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.

Different muscarinic receptors couple to different G- proteins Muscarinic Receptors: all G-protein linked M 1 : G q/11 Gastric secretion in parietal cells and depolarization of autonomic and CNS neurons M 2 : G i Negative inotropic and chronotropic effect on the heart. M 3 : G q/11 Stimulates smooth muscle contraction; sweat, bronchial and salivary secretions; paradoxical vasodilation.

Cholinergic Receptors Muscarinic Receptors M 1 Receptors G q/11 : Activation of phospholipase C generates DAG and IP 3 ; IP 3 increases intracellular calcium i.e. M 1 and  1 have similar signaling mechanism

PLC Gq M1M1 Interstitial fluid PIP 2 DAG IP 3 IP 3 R Ca 2+ GTP GDP Intracellular calcium pools    Acetylcholine Neurons: Depolarization of autonomic and CNS neurons PKC

Cholinergic Receptors Muscarinic Receptors M 2 Receptors G i : inhibition of adenylyl cyclase and decreased cAMP M 2 and  2 have similar signaling mechanism Reduced PKA phosphorylation of troponin I, negative inotropic and chronotropic effect on the heart (i.e. antagonistic to  1 )

Cardiac Muscle

Cholinergic Receptors Muscarinic Receptors M 3 Receptors G q/11 : Activation of PLC, hydrolysis of IP 3 and increased intracellular calcium, similar to M 1. 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 PGI 2 in vascular endothelium

Indirect effect of M 3 stimulation on vascular smooth muscle: vasodilation G q/11 GsGs Guanylyl cyclase Vasodilation COX – cyclo-oxygenase PCS – prostacyclin synthase M3M3 Simplistically: the endothelial cell converts a Gq response (increased Ca2+) to a Gs response (increased cyclic AMP)

Cholinergic Receptors Nicotinic Receptors: Cation Channels N M: Depolarization of NMJ; Skeletal muscle contraction. N N: Activation of post ganglionic neurons.

Cholinergic Receptors Nicotinic Receptors: Cation Channel N N Type Autonomic ganglia: Activation of post ganglionic neurons in autonomic ganglia. Agonist: Nicotine Antagonist: Trimethaphan

Binding Open K + Na + Ach N N nicotinic receptors: Heteropentamers of  and  subunits or Homopentamers of  subunits.

Cholinergic Receptors Nicotinic Receptors: Cation channel N M Type Neuromuscular Junction: Depolarization of NMJ; Skeletal muscle contraction. Heteropentamers of  and  subunits Agonist: Acetylcholine Antagonist: Tubocurarine

Nicotinic Receptor (N M )