L20 - Signalling Via Intracellular Receptors

Question 1

Why are some signalling molecules intracellular?

Answer 1

Some signal molecules are hydrophobic or small enough to pass across the plasma membrane and interact directly with intracellular enzymes or receptors

Question 2

What are two examples of signalling via intracellular receptors?

Answer 2

Nitric oxide

Lipid soluble signalling molecules e.g. retinoic acid

Question 3

Receptors bound by lipid soluble signalling molecules are?

Answer 3

Structurally related and form the nuclear receptor superfamily

Question 4

What are the two types of receptors involved in signalling via intracellular receptors?

Answer 4

Intracellular receptors

Nuclear receptors

Question 5

What are intracellular receptors?

Answer 5

Nitric oxide (NO•)
Carbon monoxide (CO)
Often have carrier proteins associated with them

Question 6

What are nuclear receptors?

Answer 6

Steroid hormones
Thyroid hormones
Retinoids
Vitamin D

Question 7

What are the 3 key properties of nitric oxide?

Answer 7

Gaseous
Free radical - unpaired electron in outer shell
Very reactive - half-life 5-10s

Question 8

What are the 3 side molecules produced in the two successive oxidations to produce nitric oxide?

Answer 8

L-arginine –> N-hydroxyarginine –> L-citrulline + NO

Question 9

What enzyme catalyses the oxidations to produce nitric oxide?

Answer 9

Nitric oxide synthase (NOS)

Question 10

What sort of expression does constitutive NOS have?

Answer 10

Expression constitutive – expressed all the time

Question 11

What is constitutive NOS induced by?

Answer 11

Enzymatic activity induced by Ca2+/calmodulin
When Ca activates them the activity is short-lived
- Produces picomoles of NO

Question 12

When activated what does constitutive NOS form?

Answer 12

Picomoles of NO

Question 13

What are the two types of constitutive NOS?

Answer 13

eNOS – endothelial

nNOS – neural

Question 14

Where is eNOS expressed?

Answer 14

Bound at cell membrane 
Endothelium 			
Cardiac myocytes			
Renal mesangial cells		
Osteoblasts/osteoclasts
Platelets

Question 15

Where is nNOS expressed?

Answer 15

Found in the cytoplasm
CNS
NANC neurons
ENS
Retina

Question 16

What is induced NOS induced by?

Answer 16

Transcriptionally induced in response to pathological stimuli - LPS, IFN-g, IL-1

Question 17

What kind of activity does inducible NOS have?

Answer 17

Enzymatic activity constitutive – active all the time

Question 18

What kind of expression does inducible NOS have?

Answer 18

Expression inducible
When induced they have a longer lasting effect
Produce nanomoles of nitric oxide

Question 19

When activated what does inducible NOS form?

Answer 19

Nanomoles of nitric oxide

Question 20

Where is inducible NOS expressed?

Answer 20

Macrophages and Kupffer cells
Neutrophils
Fibroblasts
Vascular smooth muscle and endothelial cells

Question 21

What is the effect of nitric oxide in the vasculature?

Answer 21

1. Autonomic nerves release Ach - acts on eNOS
2. eNOS release nitric oxide - causes surrounding smooth muscle to relax - vasodilation
3. NO inhibits platelet aggregation and vascular smooth muscle proliferation
   a. Anti-atherosclerotic

Question 22

How is nitric oxide induced?

Answer 22

1. Acetylcholine activates nitric oxide synthase (cNOS) in endothelial cells
2. NOS generates nitric oxide from arginine
3. Nitric oxide activates the production of cGMP

Question 23

What does nitric oxide activate the production of?

Answer 23

cGMP

Question 24

How does nitric acid activate the production of cGMP?

Answer 24

1. cGMP
2. cGMP – dependent protein kinase
3. Myosin light chain phosphatase
4. Myosin light chain

Question 25

What is the effect of nitric oxide in the brain?

Answer 25

1. In the CNS, nNOS is tethered close to NMDA-type glutamate receptors 2. This means it can respond to Ca2+ increases near the open channels a. Implications for synaptic plasticity 3. Glutamate is produced by a presynaptic terminal and this activates glutamate receptors 4. Especially NMDA receptors which stimulate the nitric oxide pathway 5. The nitric oxide produced travels to the presynaptic terminal 6. It increases the production of glutamate - creating the state of long-term potentiation

Question 26

What is the role of nitric oxide in the immune system?

Answer 26

Kills bacteria and parasites Induces programmed cell death Triggers inflammatory diseases when overproduced in lungs and intestines - Rheumatoid arthritis, Crohn’s disease and asthma Role in tumour apoptosis and necrosis

Question 27

What does iNOS in inflammatory cells use nitric oxide for?

Answer 27

Use nitric oxide as a cytostatic and cytotoxic agent | Involves much higher amounts of NO

Question 28

How was nitroglycerine use as a pharmaceutical?

Answer 28

100 yr treatment for angina - pain due to inadequate blood flow to heart Nitroglycerine breaks down in vivo to generate nitric oxide which relaxes blood vessels - Decreases load on heart - Increases heart blood supply

Question 29

What is dynamite made of?

Answer 29

Nitroglycerine + fine clay as a stabiliser

Question 30

What does nitric oxide activate in smooth muscle?

Answer 30

Guanylyl cyclase which leads to formation of cGMP

Question 31

How can too much cGMP in a cell be fixed?

Answer 31

Phospho-diesterase 5 | Converts cGMP to GMP sing cGMP dependent protein kinase

Question 32

How does Viagra work?

Answer 32

1. Inhibits phosphodiesterase 5 2. Leads to less breakdown of cGMP 3. Smooth muscles constricting blood vessels relax 4. More blood flow

Question 33

What are some examples of the nuclear receptor superfamily?

Answer 33

``` Cortisol Estradiol Testosterone Thyroxine Vitamins D3 Retinoic acid ```

Question 34

How many domains do nuclear receptors have?

Answer 34

``` 3 Transcription activating domain DNA binding domain Regulatory binding domain - Binds to ligands releasing inhibitory protein ```

Question 35

How do nuclear receptors go from their active state to inactive state?

Answer 35

1. Inhibitory proteins hold the receptors in inactive state 2. Binding to ligand removes inhibitors leading to a conformational change 3. Receptors now interact with coactivators to direct transcription The effects of the ligands are modified in different tissues by the expression of different coactivators

Question 36

What is the structural organisation of the estrogen receptor?

Answer 36

DNA binding domain consists of Zinc Fingers | Each finger binds a specific DNA sequence

Question 37

Examples of symmetric homodimers

Answer 37

Glucocorticoid and estrogen receptors

Question 38

Examples of heterodimers

Answer 38

Vitamin D, thyroid hormone, and retinoic acid receptors

Question 39

How do symmetric homodimers bind?

Answer 39

Bind as symmetric homodimers to an inverted repeat DNA site

Question 40

How do heterodimers bind?

Answer 40

Bind as heterodimers to direct repeat sites | RAR:RXR or VDR:RXR

Question 41

Where are homodimeric receptors found?

Answer 41

Anchored in the cytoplasm by inhibitor proteins in the absence of ligand - E.g. Hsp90 Hormone binding causes release of inhibitor proteins allowing entry to nucleus

Question 42

Where are heterodimaeric receptors found?

Answer 42

Located exclusively in nucleus

Question 43

How do heterodimeric receptors act in the absence of ligands?

Answer 43

Act as repressors in the absence of ligand - recruitment of histone deacetylases

Question 44

How do heterodimeric receptors act in the presence of ligands?

Answer 44

Act as activators in the presence of ligand - recruitment of histone acetylases

Question 45

Early primary response to steroid hormone – 1-6 hours

Answer 45

Steroid-hormone receptor complexes activate transcription of primary response genes Some of these genes will encode transcription factors Induced synthesis of a few proteins

Question 46

Delayed secondary response to steroid hormones – 6-48 hours

Answer 46

Primary response protein shuts of primary response genes and turn on secondary response genes Induced synthesis of secondary response proteins