PSC2002 Membrane Transport and Cell Signalling

Question 1

Name some components of the cell membrane

Answer 1

-Phospholipid bilayer
-Gprotein coupled receptor
-Channels
-Membrane proteins

Question 2

Describe Macrotransfer across a cell membrane and some examples.

Answer 2

-Large scale movement of molecules
eg -Endocytosis (membrane wraps around stuff and brings it into the cell)
-Exocytosis (vesicles fuses with membrane to release substances)

Question 3

Describe Microtransfer across a cell membrane and an example.

Answer 3

-Smaller scale movement of molecules
eg -Cell membrane transport of small molecules and ions

Question 4

What is vesicular transport involved in?

Answer 4

Endocytosis and Exocytosis through the cell surface membrane

Question 5

Describe the decreasing plasma membrane permeability of certain molecules

Answer 5

-Hydrophobic molecules
-Small uncharged polar molecules
-Large polar molecules
-Ions

Question 6

Describe the resting membrane potential of all cells

Answer 6

-Determined by distribution of ions across the membrane
-Mostly due to Na+ and K+
-Determined by the Nernst equation

Question 7

Outline the Nernst equation

Answer 7

Em = (RT / zF) * ln([ion conc outside] / [ion conc inside])

Em = Membrane potential
RT = Gas constant x Temperature
zF = Number of charges on ion x Faraday’s number

Question 8

What does the Nernst equation predict?

Answer 8

It predicts equilibrium membrane potential based on concentration gradient of that ion across that membrane

Question 9

Describe the permeability ratio of K+:Na+ in Non excitable cells and nerve/muscle cells.

Answer 9

Non-excitable cells 2:1
Nerve/Muscle cells 25:1

Question 10

Describe how a voltage clamp works

Answer 10

-A specific membrane potential (voltage) is set
-The clamp measures how much current (ionic flow) is needed to keep the membrane potential al that set value
-Despite natural changes in ion concentration and permeability

Question 11

What channel properties can a voltage clamp tell us?

Answer 11

-Activation and inactivation kinetics in response to voltage changes
-Reversal potential (MP at which there is no net ionic current through a particular ion channel, indicating the ion’s equilibrium potential)
-Pharmacological effects

Question 12

What are the main applications of the voltage clamp to study membrane potential?

Answer 12

-Ion channel analysis
-Pharmacology studies

Question 13

What are the main applications of the current clamp to study membrane potential?

Answer 13

-Action potential studies
-Excitability studies

Question 14

Describe how current clamp works

Answer 14

-A constant or variable current is injected into the cell
-The resulting changes in MP are measured
-Indicating how the cell responds to the injected current (including action potential firing, threshold potential)

Question 15

Describe how patch clamping works

Answer 15

-Involves using a micropipette to form a high resistance seal with the cell membrane, allowing for measurement of ionic currents
-Can isolate the activity of individual ion channels

Question 16

Describe the method by which molecules move directly across the lipid bilayer

Answer 16

-Passive (simple) diffusion
-Solute/gas passes down concentration gradient
-At finish inward flux = outward flux
-Obeying Fick’s law of diffusion

Question 17

What methods of moving across the phospholipid bilayer are there using integral membrane proteins

Answer 17

-Facilitated diffusion (through pore, channels and carriers)
-Active transport (requiring ATP to transport)
-Secondary active transport

Question 18

What are the types of secondary active transport?

Answer 18

-Co-transport (Symporters) involve movement of a solute coupled to the movement of another down its concentration gradient
-Counter-transport (antiporters) involve coupled movement of two or more solutes in opposite direction

Question 19

Describe Fick’s law

Answer 19

Flux = Permeability x concentration difference

Question 20

How do you determine permeability?

Answer 20

(Diffusion coefficient x Partition coefficient) / Distance

Question 21

What are the three types of transporters that facilitate diffusion?

Answer 21

-Channels (non gated)
-Gated channels
-Uniporters (carriers)

Question 22

Name some important integral non gated channels

Answer 22

-Porins in bacteria, mitochondria and nuclear pore complex
-Aquaporins

Question 23

What is ENaC?

Answer 23

Epithelial sodium channel

Question 24

Name some important gated channels

Answer 24

-ENaC
-K+ channels
-Ca2+ channels
-Almost all ion channels

Question 25

Give some functional components of gated channels

Answer 25

-Gate -Sensor (detects signal to open gates) -Selectivity filter (stops other substances)

Question 26

What modulates the function of gated channels?

Answer 26

-Voltage -Mechanical stimuli -Ligand binding

Question 27

Name some important uniporters (carrier channels)

Answer 27

-GLUT1 (RBC) -GLUT2 (Intestines) -GLUT4 (Skeletal/cardiac muscle)

Question 28

Describe the cycle by which uniporters regulate movement through a membrane

Answer 28

-Carrier open -Solute enters and binds -Outer gate closes -Inner gate opens -Solute released -Inner gate closed

Question 29

What can inhibit transporters?

Answer 29

Structural analogues

Question 30

Describe how the Na+/K+ pump is electrogenic

Answer 30

-It drives 3 Na+ ions out for every 2 K+ ions in -Contributing 10% electrical potential across cell membrane

Question 31

Name some examples of P-type ATPases

Answer 31

-Na+/K+ pumps -Ca2+ pumps in SR

Question 32

What can be used to inhibit the Na+/K+ pump

Answer 32

-Ouabain -Cardiac glycosides

Question 33

Name the types of domains found in the Na+/K+ pump

Answer 33

Intra, trans and extra membrane domains

Question 34

Name the intracellular domains found in the Na+/K+ pump

Answer 34

-Actuator domain -Phosphorylation domain -Nucleotide domain

Question 35

Describe the binding sites for Na+ and K+ in the Na+/K+ pump

Answer 35

-3 binding sites -However K+ ions are larger so Na+ fits into domains easier -Leading to the 3:2 stoichiometry

Question 36

Describe the mechanism in the Na+/K+ pump

Answer 36

-Na+ enters the channel and causes conformational change to move Nucleotide domain across -Nucleotide domain phosphorylates P domain, releasing ADP and energy, which issued to shut gate on one side and open gate on other side -Na+ is released, and K+ enters the now vacant binding site in channel, causing conformational change to release phosphate from P domain through the actuator domain -Regenerated ATP binds N domain, and energy is used to open gate, causing K+ to exit and Actuator to reset

Question 37

Describe the Gibbs-Donnan effect

Answer 37

-Describes how the presence of impermeable ions influence the distribution of other ions across that membrane. -Permeable ions will distribute to balance concentration gradients and electrostatic forces.

Question 38

Why is the Na+/K+ pump needed to counteract the Gibbs-Donnan effect

Answer 38

Na+/K+ pump forces positive ions out of the cell, increasing the water potential, preventing too much osmotic pressure (ie water entering the cell and lysing)

Question 39

What do secondary active transporters use to transport substances

Answer 39

-Use the kinetic energy provided by electrochemical gradients as -One solute is transported down a concentration gradient -With another solute coupled to this transport

Question 39

Describe symporters

Answer 39

Secondary active transporters involved in the transport of two or more molecules in the same direction

Question 40

Give an example of a symporter

Answer 40

Na+/Glucose transporter (SGLT)

Question 41

Describe antiporters

Answer 41

Secondary active transporters involved in the transport of two or more ions in opposite directions

Question 42

Give examples (and their functions) of antiporters

Answer 42

-NHE (Na+/H+) maintains cytosolic pH -NCX (Na+/Ca2+) maintains low cytosolic Ca2+ conc

Question 43

What are ABC transporters?

Answer 43

ATP Binding Cassette transporters

Question 44

Describe ATP Binding Cassette transporters

Answer 44

-Utilise energy from ATP hydrolysis to transport various molecules across cellular membrane -Can remove toxins

Question 45

Give an example of an ATP Binding Cassette transporter

Answer 45

CFTR

Question 46

How is CFTR an irregular ATP Binding Cassette transporter?

Answer 46

As ATP regulates the channel

Question 47

Describe the domains found in ABC transporters

Answer 47

-Two Membrane spanning domains -Two nucleotide binding domains -One Regulatory domain

Question 48

What is Osmosis

Answer 48

Passive movement of a solvent to a region of high solute concentration across a semi-permeable membrane

Question 49

What are the major solutes driving osmosis?

Answer 49

IONS

Question 50

What is osmolarity and its units

Answer 50

Total concentration of dissolved particles in a litre of solution (osmol/L)

Question 51

What is osmolality and its units

Answer 51

The number of dissolved particles per unit mass (osmol/kg H2O)

Question 52

What is osmotic pressure?

Answer 52

Pressure exerted by flow of water across membrane, determined by solute concentrations

Question 53

What is tonicity

Answer 53

A measure of the effect a solution has on cells placed in it and is driven by osmolarity

Question 54

Describe a Hypotonic solution

Answer 54

-Has an osmolarity less than intracellular -Water moves into the cell and it expands

Question 55

Describe a Hypertonic solution

Answer 55

-Has an osmolarity greater than intracellular -Water moves out of the cell and it shrinks

Question 56

Describe an isotonic solution

Answer 56

-Has an osmolarity that equals intracellular -No net movement and no change in cell size

Question 57

Describe the equation to find water flow

Answer 57

Water flow = Hydraulic water permeability x Pressure

Question 58

What protein is involved in the transport of water across a membrane?

Answer 58

Aquaporins

Question 59

If a solute reflection coefficient (σ) is 1, how permeable is the membrane?

Answer 59

Absolutely impermeable

Question 60

If a solute reflection coefficient (σ) is 0.5, how permeable is the membrane?

Answer 60

Semi permeable

Question 61

If a solute reflection coefficient (σ) is 0, how permeable is the membrane?

Answer 61

No barrier, it is fully permeable

Question 62

Describe Epithelial tissues

Answer 62

-Line external and internal surfaces -Internal is known as endothelium, external as epithelium

Question 63

What lines the lumen of the gut?

Answer 63

Epithelial cells making up the epithelium

Question 64

Where is epithelia found?

Answer 64

-Line outside of human body (skin) -Line internal cavities and lumen of bodies

Question 65

Give some prominent examples of epithelia

Answer 65

-GI tract -Genito-urinary tract -Respiratory tract

Question 66

How many human cancers originate from epithelial cells?

Answer 66

85%

Question 67

Give some functions of epithelia

Answer 67

-Protection -Filtration (lining of kidney tubules) -Exchange (alveoli) -Absorption (intestine) -Sensation (taste buds, olfactory epithelium) -Secretion (lining of glands)

Question 68

Give some different epithelial tissue types

Answer 68

-Simple squamous -Simple cuboidal -Simple columnar -Stratified squamous -Pseudostratified columnar -Transitional

Question 69

Give the function and location of simple squamous epithelial tissue

Answer 69

FUNCTION - Absorption, filtration, minimal barrier to diffusion LOCATION - Capillaries, alveoli, abdominal and pleural cavities

Question 70

Give the function and location of simple cuboidal epithelial tissue

Answer 70

FUNCTION - Secretion, transportation LOCATION - Glands and ducts, kidney tubules, ovaries

Question 71

Give the function and location of simple columnar epithelial tissue

Answer 71

FUNCTION - Absorption, protection, secretion LOCATION - Digestive tract

Question 72

Give the two types of simple columnar epithelial tissue

Answer 72

Ciliated and non-ciliated

Question 73

Give the function and location of stratified squamous epithelial tissue

Answer 73

FUNCTION - Protection LOCATION - Skin, Mouth, Upper throat, Oesophagus

Question 74

Give the two types of stratified squamous epithelial tissue

Answer 74

-Keratinised (makes impermeable and dry) -Non-keratinised (kept moist by secretions to prevent from drying out)

Question 75

Give the function and location of pseudostratified columnar epithelial tissue

Answer 75

FUNCTION - Absorption and protection LOCATION - Upper respiratory tract, trachea

Question 76

Give the function and location of transitional epithelial tissue

Answer 76

FUNCTION - Stretchable layer LOCATION - Bladder

Question 77

How do epithelia form a functional unit

Answer 77

-Epithelial layers are attached to the extracellular matrix of the basal lamina -Linked using specialised structures

Question 78

What structures link epithelial cells to each other and the basal lamina?

Answer 78

-Tight junctions -Anchoring junctions -Channel forming junctions

Question 79

Describe tight junctions

Answer 79

-Membrane proteins that seal adjacent cells together -Ensuring molecules cannot leak freely between cells -Also prevent lateral migration of membrane proteins

Question 80

By preventing lateral migration of membrane proteins, what do tight junctions in epithelial cells generate?

Answer 80

Cell polarity, as distinct cell membrane domains are created

Question 81

What controls the flow of molecules into epithelial cells?

Answer 81

The paracellular barrier, which is established by tight junction proteins

Question 82

Give an example of an epithelium containing a high vs a low number of tight junction proteins

Answer 82

High - Urinary bladder Low - Proximal tubule

Question 83

What combination of Claudins that make up a tight junction protein make it tight?

Answer 83

Claudin-1 and Claudin-3

Question 84

What combination of Claudins that make up a tight junction protein make it leaky?

Answer 84

Claudin-1 and Claudin-2

Question 85

Describe anchoring junctions found in epithelia

Answer 85

-Provide mechanical stability -Anchor cells to the basal lamina, or to other cells -Two categories

Question 86

What are the two categories of anchoring junctions in epithelia?

Answer 86

-Actin attachments -Intermediate filament attachments

Question 87

What are the two types of actin attachment anchoring junctions in epithelia?

Answer 87

-Cell to cell: Adherens junction -Cell to basal lamina: Focal adhesions

Question 88

What are the two types of intermediate filament attachment anchoring junctions in epithelia?

Answer 88

-Cell to cell: desmosomes -Cell to basal lamina: hemidesmosomes

Question 89

Describe the basal lamina

Answer 89

Strong and flexible foundation that underlies all epithelia

Question 90

How thick is the basal lamina?

Answer 90

40-120 nm thick

Question 91

Name the interactions that form the meshwork of the basal lamina

Answer 91

-Laminin -Type IV collagen -Entactin -Perlecan

Question 92

Describe the basement membrane

Answer 92

-Combination of the basal lamina and the reticular lamina -Anchors epithelial cells to connective tissue below -Acts as a mechanical barrier -Important in angiogenesis

Question 93

What is the function of gap junctions in epithelial cells?

Answer 93

-Form intracellular channels (approx 1.5nm in diameter) -Allowing diffusion between cells and enabling cell to cell communication

Question 94

What are the functions of Ussing chambers?

Answer 94

-Measures resistance of ion transport -Used to determine what ion channels are present (and their concentration and expression)

Question 95

Describe Ussing studies of frog skin

Answer 95

-Skin dissected and mounted as flat sheet between two chambers containing solution of identical composition -Skins developed a trans epithelial potential difference due to "active" transepithelial Na+ movement

Question 96

What do polarised epithelial cells generate?

Answer 96

Transepithelial voltage due to a total difference in charge across the cell (basolateral - apical)

Question 97

Describe the mechanism by which epithelial NaCl and water is absorbed

Answer 97

-Na+ influxes through ENaC -Na+ is actively pumped out of the cells across the basolateral membrane via the Na+/K+ ATPase -Causing paracellular transport of Cl via tight junctions to maintain electroneutrality -Increasing the NaCl conc on the basolateral side of the epithelium, driving osmotic movement of water

Question 98

Name some epithelial tissues containing ENaC

Answer 98

-Kidney -Lung -Colon -Sweat gland

Question 99

Simply, what is the role of ENaC in the kidney?

Answer 99

Na+ retention, controlling whole body Na+ and water balance, and therefore blood pressure

Question 100

What is the role of ENaC in the lung?

Answer 100

Na+ and water absorption, controlling the amount of airway surface liquid (ASL) and alveolar lining fluid (ALF)

Question 101

What is the role of ENaC in the colon?

Answer 101

Na+ and water reabsorption from the diet

Question 102

What is the role of ENaC in the sweat gland?

Answer 102

Na+ retention, reabsorption of Na+ by sweat ducts, but this is not followed by water, producing a hypotonic sweat secretion

Question 103

Describe the degenerin ion channel family, and give an example

Answer 103

-"Acid-sensing" -eg ENaC

Question 104

Describe the structure of ENaC

Answer 104

-Functional channel is a heterotrimer of 3 ⍺, β or γ subunits, coded by 3 genes -Each subunit has 2 transmembrane domains (forming the pore) -Extracellular loops are the site for regulation by CAPs and SPLUNC1

Question 105

What may regulate ENaC?

Answer 105

CAPs and SPLUNC1

Question 106

What is the site for ubiquitination in ENaC?

Answer 106

PY motif in C term

Question 107

How does aldosterone affect final urinary salt composition?

Answer 107

-Changes ENaC function in the aldosterone sensitive distal nephron -Binds to cytosolic mineralocorticoid receptor, which goes to nucleus and binds with HRE genes, increasing SGK

Question 108

What occurs in principal cells of the aldosterone sensitive distal nephron following stimulation by HRE?

Answer 108

-Surface ENaC levels increase by 2-5 fold -Increasing Na+/K+ ATPase density -increased ATP supply to support increased Na+/K+ ATPase activity -Increased K+ excretion across apical membrane via ROMK

Question 109

Where is the Aldosterone sensitive distal nephron located?

Answer 109

In the last third of the distal convoluted tubule (DCT2), the connecting tubule (CNT) and the cortical collecting duct (CCD)

Question 110

How does aldosterone increase the number of ENaC in the apical membrane?

Answer 110

-ALDOSTERONE decreases the rate of retrieval by stimulating SGK1, which phosphorylates NEDD4 preventing it binding to the PY motif and inhibiting ENaC ubiquitination -Preventing internalisation

Question 111

What may be the result of excess aldosterone?

Answer 111

Hypertension, hypokalaemia and or alkalosis

Question 112

What is Liddle's syndrome characterised by?

Answer 112

-Mutations in cytoplasmic region of β or γ ENaC -Leading to hypertension, hypokalaemia and or alkalosis

Question 113

Which genes are mutated in Liddle's syndrome?

Answer 113

SCNN1A, SCNN1B and SCNN1G

Question 114

How do the mutations caused by Liddle's syndrome lead to excessive gain of function in ENaC?

Answer 114

Mutations lead to changes in cytoplasmic region of ENaC subunits, preventing NEDD4-2 binding, increasing the number of ENaC

Question 115

Describe how increased ENaC function causes hyperkalemia in Liddle's syndrome

Answer 115

-Enhanced ENaC function depolarises apical membrane potential -Electrical gradient for K+ excretion through ROMK is increased so get excessive loss of K+ in the urine leading to low blood K_

Question 116

Describe the effect of Amiloride

Answer 116

-Inhibits ENaC (preventing Na+ transport), lowering blood pressure -Hyperpolarises the apical membrane potential, reducing K+ loss and preventing hypokalaemia

Question 117

Where is ENaC expressed in the lungs?

Answer 117

-In the apical membrane of surface epithelial and alveolar type II cells -ie conducting and respiratory airways

Question 118

What is airway surface liquid (ASL) vital for?

Answer 118

Innate defence of the lungs via mucociliary clearance

Question 119

What is alveolar lining fluid (ALF) vital for?

Answer 119

Efficient gas transfer

Question 120

What mediates ENaC in the conducting airways?

Answer 120

↑Channel activating proteases (CAPS) ↓Antiproteases ↓SPLUNC1 ↓ATP

Question 121

Describe the different ways in which each NaCl ion is secreted by epithelial cells

Answer 121

Na+ moves paracellularly, Cl- moves transcellularly

Question 122

What are the two types of chloride ion channels?

Answer 122

CFTR and Calcium Activated Chloride Channel (CaCC)

Question 123

What differentiates CFTR from other ABC transporters?

Answer 123

It is an ion channel, not an active pump

Question 124

What domains make up CFTR?

Answer 124

5 -MSD1 and MSD2 (forming the pore) -NBD1 and NBD2 (bind ATP) -R domain (PKA phosphorylation site)

Question 125

Describe the molecular mechanism of CFTR gating

Answer 125

-PKA phosphorylation of the R domain induces ATP binding and dimerisation of the NBDs -Conformational change in NBDs transmitted to MSDs, leading to pore opening -ATP is hydrolysed and pore closes -Dephosphorylation by protein phosphates closes the channel

Question 126

What has been used to demonstrate that CFTR requires both PKA phosphorylation and ATP binding to open?

Answer 126

Inside out patch

Question 126

What may modulate Calcium activated Chloride channels (CaCC)?

Answer 126

Calmodulin (CaM) and CAM dependent Kinase (CaMK)

Question 127

Describe where Calcium activated Chloride Channels (CaCC) are found

Answer 127

-Present in the apical membrane of most epithelial cells expressing CFTR (apart from intestinal cells) -Also found in apical membrane of gland secretory acinar cells (WITH NO CFTR) -In some endocrine, smooth and skeletal muscle, and neurones

Question 128

What activates Calcium activated Chloride channels (CaCC)?

Answer 128

A rise in cytosolic Ca2+

Question 129

What are TMEM16A and TMEM16B?

Answer 129

Calcium activated Chloride Channels

Question 130

Describe the structure of TMEM16A (CaCC)?

Answer 130

-Contains 10 TMDs, with the pore region being TMD 6-9 -Contains 3 intracellular loop -Functional channel is made up of TMEM16A dimer

Question 131

Describe how TMEM16A is activated

Answer 131

-Calcium ions bind to glutamate residues in one of the two ⍺ helices of intracellular loop 3 -Causing ⍺ helices to move apart, opening the pore, enabling chloride transport

Question 132

What are the two main mechanisms by which HCO3- is secreted?

Answer 132

-Directly through the chloride channel itself -Indirectly via coupling the chloride channel with an apical chloride/bicarbonate exchanger

Question 133

What apical anion exchanger is involved in NaHCO3 secretion?

Answer 133

SLC26A, a Cl-/HCO3- exchanger

Question 134

Describe how a net NaHCO3 yield secretion occurs in the epithelia?

Answer 134

A chloride channel is coupled to SLC26A

Question 135

As well as containing SLC26A, what else do many epithelia that secrete HCO3- have?

Answer 135

Na+ dependent HCO3- cotransporters (NBCs) on the basolateral membrane, supplying cytosolic HCO3- for the SLC26A exchanger

Question 136

How does PKA phosphorylation of CFTR influence other channels?

Answer 136

-Switches on anion exchanger activity for SLC26A -By physically interacting via the scaffold protein CAP70

Question 137

Which epithelial tissues does HCO3- secretion occur in (requiring regulation by CFTR)?

Answer 137

-Small intestine -Biliary tract -Exocrine pancreas -Airways -Female and Male reproductive tracts

Question 138

Which epithelial tissues does HCO3- secretion occur in (not requiring regulation by CFTR)?

Answer 138

The stomach

Question 139

Describe acinar cells in the exocrine pancreas

Answer 139

Secrete a variety of digestive enzymes and a low volume, NaCl rich fluid into the ducts, using TMEM16A channels

Question 140

Describe duct cells in the exocrine pancreas

Answer 140

Transport digestive enzymes to small intestine. Produce a high volume, NaHCO3 rich secretion using both CFTR and SLC26A6

Question 141

What is fluid secretion in the GI tract driven by?

Answer 141

Both NaCl and NaHCO3 secretion with CFTR as the dominant anion channel

Question 142

What mechanisms carry out fluid absorption in the GI tract?

Answer 142

-ENaC mediated fluid absorption in the colon -Sodium linked absorptive ion transporters (SLC26A, NHE3) -Nutrient absorptive transporters (SGLT1, GLUT5)

Question 143

Describe how vibrio cholerae infection causes secretory diarrhoeas

Answer 143

Cholera toxin inhibits NaCl and fluid absorption (from villi) and stimulates CFTR mediated Cl-/HCO3- and fluid secretion (from crypts)

Question 144

Describe how Cholera toxin overstimulates CFTR?

Answer 144

CT causes ADP ribosylation of G protein that blocks GTP hydrolysis by the subunit, becoming permanently active (producing lots of cAMP)

Question 145

What may be given to treat secretory diarrhoeas?

Answer 145

-Oral Rehydration Therapy -Isomolar or hypoosmolar salt solution containing NaCl, NaCitrate, KCl and glucose

Question 146

What pattern of inheritance describes Cystic fibrosis?

Answer 146

Autosomal recessive

Question 147

What is cystic fibrosis caused by?

Answer 147

Loss of function mutations in the Cystic Fibrosis Transmembrane Conductance regulator (CFTR) gene

Question 148

How many people in the UK live with CF?

Answer 148

Over 11,000

Question 149

How many people in the UK are carriers for cystic fibrosis?

Answer 149

1 in 25

Question 150

How many mutations in the CFTR gene cause CF?

Answer 150

720

Question 151

What is the most common mutation in the CFTR gene leading to CF?

Answer 151

-F508del, loss of phenylalanine at position 508 in NBD1 -50% are homozygous for F508del, and 35% contain a single F508del allele

Question 152

What are the two types of CFTR mutations?

Answer 152

-Minimal function mutations -Residual function mutations

Question 153

What are the different classes of CFTR mutations for those with CF?

Answer 153

-No protein (class I) -No traffic (class II) -No function (class III) -Less function (class IV) -Less protein (class V) -Less stable (class VI)

Question 154

Give an example of a class I CFTR mutation

Answer 154

G542X

Question 155

Give an example of a class II CFTR mutation

Answer 155

F508del

Question 156

Give an example of a class III CFTR mutation

Answer 156

G551D

Question 157

Give an example of a class IV CFTR mutation

Answer 157

R117H

Question 158

Give an example of a class V CFTR mutation

Answer 158

A455E

Question 159

Give an example of a class VI CFTR mutation

Answer 159

rF508del

Question 160

Which classes of CFTR mutations produce the most severe phenotype?

Answer 160

Classes I, II and III ie Minimal function mutations

Question 161

Can Cystic fibrosis phenotype severity be predicted from genotyping?

Answer 161

-Sometimes, depending on which organ -Lung disease severity is more variable (influenced by environment, other genetic modifiers) -Pancreatic insufficiency can correlate well with genotype

Question 162

How does cystic fibrosis affect the function of the pancreas?

Answer 162

-Defective CFTR leads to reduced chloride and bicarbonate secretions, causing sticky mucus that block ducts from acinar cells -Leading to -acidic environment which activates digestive enzymes, digesting the ducts -blocked enzyme flow to the small intestine

Question 163

What is the role of CFTR in conducting airways?

Answer 163

-Maintains proper hydration and pH of the airway surface liquid (ASL), which induces the PCL layer and the mucus layer -Ensuring efficient mucociliary clearance

Question 164

Name some cell types in the conducting airway epithelium

Answer 164

-Club cell -Goblet cell -Basal cell -Ciliated cell -Ionocyte -PNEC -Tuft cell

Question 165

How does cystic fibrosis affect mucociliary clearance?

Answer 165

-Airway surface liquid is dehydrated and more acidic leading to viscous mucus, mucus accumulation and obstruction, and failure of MCC -Leading to chronic bacterial colonisation and the establishment of biofilms -Leading to destruction as chronic immune response in this area

Question 166

How does cystic fibrosis lead to more acidic airway surface liquid?

Answer 166

-Lack of CFTR function reduces HCO3- secretion into the ASL -H+ secretion is active, producing more acidic ASL

Question 167

What are the consequences of a more acidic ASL?

Answer 167

-Increases fluid absorption due to enhanced ENaC activity (increased CAP and decreased SPLUNC1) -Increased mucus stasis and viscosity (decreased mucin release) -Decreased bacterial killing (reduced AMP activity)

Question 168

What are the common symptoms that characterise cystic fibrosis in the lungs?

Answer 168

-Mucus clogging -Reduced airway surface liquid -Recurrent lung infections -Overactive immune response -Destruction of lung

Question 169

How is mucus clogging in the lungs in cystic fibrosis treated?

Answer 169

Using physiotherapy and mucolytics

Question 170

How is reduced airway surface liquid in the lungs in cystic fibrosis treated?

Answer 170

-Using nebulised hypertonic saline or mannitol -Drawing water from the body into the airways, improving hydration

Question 171

How is overactive immune response in the lungs in cystic fibrosis treated?

Answer 171

Using NSAIDs

Question 172

What are the main approaches to the treatment of the basic anion permeability defect in cystic fibrosis?

Answer 172

-CFTR modulator therapy -Genetic therapy -Alternate channel therapy

Question 173

In terms of CFTR function, how much function is required to reduce symptoms?

Answer 173

<5% is involved in lung disease, meaning giving little CFTR function improves quality of life significantly!

Question 174

How may we assess cystic fibrosis therapies in patients?

Answer 174

-Lung function tests eg FEV1 -Sweat chloride test -Other relevant parameters such as BMI, quality of life, number of hospitalisations

Question 175

Give some key structures found in a sweat gland

Answer 175

-Secretory coil secretes NaCl rich isotonic from acinar cells (mostly Cl secretion via TMEM16A) -Absorptive duct absorbs NaCl transcellularly (but no water) producing a hypotonic fluid, using ENaC and CFTR

Question 176

What regulates ion and fluid transport in healthy sweat glands?

Answer 176

Via ACh and Catecholamines

Question 177

Describe the sweat test for Cystic fibrosis severity?

Answer 177

-Sweat glands are stimulated and sweat is collected for 30 minutes -Sweat chloride greater than 60mmol/Ln indicates Cystic fibrosis -As NaCl is not reabsorbed in absorptive duct due to faulty CFTR

Question 178

Describe CFTR modulator therapies

Answer 178

-Uses drugs to correct mutant CFTR -Can be standalone or used in combination with genetic therapies or alternate channel therapy -Can be personalised

Question 179

What aspect of CFTR activity can be altered using drug therapies?

Answer 179

-Increase the number of CFTR channels at cell surface (class II mutation) -Enhance CFTR channel gating (class III mutation) -Increase ion flux (class IV mutation)

Question 180

What are the CFTR drug modulator types that are currently in clinical use?

Answer 180

-Potentiators -Correctors

Question 181

What are the CFTR drug modulator types that are currently in development?

Answer 181

-Termination suppressors -Amplifiers -Stabilisers

Question 182

Describe what CFTR drug modulator "potentiators" do, and give an example.

Answer 182

-Increase the activity of class III gating mutants and some residual function mutants -Vertex VX770 Kalydeco or Ivacaftor

Question 183

How do CFTR drug potentiators work?

Answer 183

-By increasing the opening rate or duration of openings -eg VX770 increases G551D CFTR channel activity, and has demonstrated an increase in FEV1 and decrease in sweat chloride tests

Question 184

Describe what CFTR drug modulator "correctors" do, and give an example.

Answer 184

-Promote processing of class II mutants to the plasma membrane, increasing folding efficiency of the channel in the ER -This increases the number of channels -VX809 lumacaftor

Question 185

What have Ussing chambers demonstrated with VX809?

Answer 185

VX809 increases F508del CFTR at the apical plasma membrane of human airway cells in vitro, increasing chloride secretion following a cAMP agonist

Question 186

What defects need to be corrected in F508del CFTR?

Answer 186

-Processing defect (low Number) -Gating defect -Shorter resident time in plasma membrane (stability)

Question 187

How do we correct all the defects in F508del CFTR, in order to treat those with CF?

Answer 187

-Combination therapy of potentiator (VX770) with corrector (VX809) -or triple therapy of 2 correctors and 1 potentiator

Question 188

What is the issue with the current clinical treatments for cystic fibrosis?

Answer 188

-They only treat symptoms -Do not prevent the gradual loss in lung function in most people with cystic fibrosis

Question 189

Describe what termination suppressor drug modulators for CFTR aim to do?

Answer 189

-Work by suppressing nonsense mutations inducing a stop codon -PTC124 is one currently under development

Question 190

Describe what amplifier drug modulators for CFTR aim to do?

Answer 190

-Work by enhancing stability and transaction of CFTR mRNA -PTI428 is one currently under development

Question 191

Describe what stabiliser drug modulators for CFTR aim to do?

Answer 191

-Work by increasing the functional stability of CFTR protein in the cell membrane by anchoring it

Question 192

Define genetic therapies

Answer 192

Therapeutic approaches that -Deliver copies of the healthy gene using gene addition (DNA or mRNA) -Fix the chromosomal DNA using genome editing (eg CRISPR/Cas9)

Question 193

What vectors may be used for genetic therapies for cystic fibrosis?

Answer 193

Viral or liposome vectors

Question 194

Describe experimental gene therapies using LIPOSOME vectors for cystic fibrosis

Answer 194

-Vesicles with lipid bilayers (coupled with cations) encapsulate plasmid DNA containing functional CFTR gene -Internalised and gene is inserted into DNA and expressed

Question 194

Describe experimental gene therapies using VIRAL vectors for cystic fibrosis

Answer 194

-Uses adenovirus vector -Internalised in endosome, and corrected CFTR gene is inserted and expressed in the cell

Question 195

What are the main issues surrounding gene therapies for cystic fibrosis?

Answer 195

-Delivery challenges (patients have thick mucus that hinders delivery of vectors) -Only transient expression (requires repeated treatments) -Immune responses lead to reduced efficacy and damage associated with repeated treatments -Difficult to target correct cells -May disrupt other genes if integrated into DNA

Question 196

How may we improve the efficacy of gene therapies?

Answer 196

-Using lentivirus, AAV for long term stable correction as the gene is integrated into DNA -Modify virus to make them less immunogenic -Use other non viral approaches such as nanoparticles

Question 197

Describe the steps involved in cell therapy for cystic fibrosis

Answer 197

-Isolate cells from skin or other tissue -Generate induced pluripotent stem cells -Correct the CFTR mutation to wild type -Differentiate the cells to basal airway stem cells -Engraft onto the basal membrane

Question 198

Describe mRNA mediated therapy for cystic fibrosis

Answer 198

-Focuses on delivering synthetic messenger mRNA encoding the functional CFTR to cells -Bypassing the translation step involved in gene therapies

Question 199

Describe Antisense Oligonucleotide (ASO)-mediated therapy for cystic fibrosis

Answer 199

Focuses on binding to CFTR mRNA, suppressing defective mRNA and improving functionality of the translated CFTR protein

Question 200

What is the focus of alternate channel therapy for cystic fibrosis?

Answer 200

-Focuses on alternative chloride Chanels that are present in CF cells to bypass defective CFTR and restore Cl-/HCO3- and fluid transport -Aims to inhibit ENaC to help reduce salt and fluid absorption

Question 201

What is the main target for using alternative chloride channels to treat cystic fibrosis?

Answer 201

-TMEM16A -Need a drug that directly activates the channel without involving Ca2+

Question 202

How may we decrease ENaC activity to treat cystic fibrosis as part of "alternative channels" therapy?

Answer 202

-Use amiloride like drugs -Inhibit proteases that activate ENaC -Target ENaC regulatory proteins

Question 203

What are the two main transporter superfamilies?

Answer 203

-ATP binding cassettes (ABC) -Solute carrier (SLC)

Question 204

Name some important members of the solute carrier (SLC) transporter superfamily?

Answer 204

-OAT (Organic anion transporter) -OATP (organic anion transporting polypeptide) -OCT (Organic cation transporter) -MATE (multidrug and toxin extrusion protein)

Question 205

Do ATP binding transporters import or export?

Answer 205

-They can do either -But never both

Question 206

Name some ABC efflux transporters

Answer 206

MDR1/P-gp

Question 207

Name some ABC influx transporter

Answer 207

-Maltose uptake transporter -Methionine uptake transporter -Typically found in prokaryotes

Question 208

Name some ABC associated with ion channels

Answer 208

-CFTR -SUR1

Question 209

Where are ABCs typically expressed?

Answer 209

In cells with excretory or barrier functions, eg -Liver, intestine, kidney -Blood brain barrier, Blood placenta barrier, Blood testis barrier

Question 210

What characterises most cells expressing ABCs

Answer 210

-Polarised, with ABC transporters being expressed either on the apical or basolateral side -BUT NOT BOTH

Question 211

What do ABCs protect cells against?

Answer 211

Typically against xenobiotics

Question 212

How many genes in humans are there for ABC transporters?

Answer 212

48

Question 213

How many ABC subfamilies are there in humans?

Answer 213

7, A-G

Question 214

How many domains are there in ABC transporters?

Answer 214

-2 Nucleotide binding domains which bind and hydrolyse ATP -2 Transmembrane domains which bind and transport substrates

Question 215

How conserved are ABC NBDs and TMDs?

Answer 215

-NBDs are highly conserved -TMDs are less conserved

Question 216

Name the roles of ABC transporters in the intestine

Answer 216

-ABCs interfere with drugs entering enterocytes through brush border membrane -Pumping compounds such as drugs back into the lumen -Preventing them reaching the hepatic portal vein

Question 217

Name the roles of ABC transporters in the liver

Answer 217

-ABCs transport drugs into hepatocytes from circulation across sinusoidal membrane -ABCs transport drugs across canalicular membrane to bile or across sinusoidal membrane back to blood (for renal excretion) following metabolism

Question 218

Name the roles of ABC transporters in the blood brain barrier

Answer 218

ABC transporters at blood brain barrier prevent many potentially toxic compounds reaching brain

Question 219

Describe what ABC knockout mice studies found?

Answer 219

Drug accumulation and toxicity in the brain as they are able to cross the blood brain barrier

Question 220

Describe how a multi-drug resistant tumour is developed

Answer 220

-Primary tumour contains mostly normal cells and a few MDR cells -Following treatment, all normal cells are killed, only MDR cells remain -Relapse occurs as these MDR cells grow

Question 221

Give examples of mechanisms of multidrug resistance in cancer

Answer 221

-Decreased uptake by cell -Increased metabolism of drug in cell -Alteration in cell target -Enhanced drug efflux

Question 222

By what mechanisms may cancer cells enhance drug efflux?

Answer 222

By expressing more ABC transporters

Question 223

What is P-glycoprotein also known as?

Answer 223

MDR1

Question 224

What gene codes for P-glycoprotein/MDR1?

Answer 224

ABCB1 gene on chromosome 7

Question 225

Describe the structure of P glycoprotein

Answer 225

TMD1 attached to NBD1, which is attached to TMD2, which is attached to NBD2

Question 226

Describe the proposed model of P glycoprotein/MDR1 mediated transport

Answer 226

-Substrate binds to binding site, and ATP binds to NBD -Conformational change causes drug efflux and release of ADP -ATP is hydrolysed and conformational reset

Question 227

What is the role of P glycoprotein/MDR1?

Answer 227

Removal of xenobiotics from cells

Question 228

Describe methods of P glycoprotein/MDR1 removing xenobiotics from cells

Answer 228

-Pumps out xenobiotics from enterocytes following initial absorption -Transports xenobiotics into bile across canalicular membrane -Prevents access of many xenobiotics to brain -Transports xenobiotics into lumen of kidney on brush border membrane

Question 229

Where is P glycoprotein/MDR1 located?

Answer 229

Expressed in cells with excretory functions (liver, intestine, kidney) or barrier functions (BBB, Blood placenta barrier, Blood testis barrier)

Question 230

What are the effects of P glycoprotein/MDR1 in tissues?

Answer 230

-Limited drug absorption -Active drug elimination -Limited drug distribution into tissues

Question 231

What have Schinkel AH et al demonstrated with P glycoprotein/MDR1?

Answer 231

-Knockout studies in mice -Viable, fertile and phenotypically normal -Suffer toxicity with some compounds due to entry into brain -Show increased absorption and decreased excretion of a number of drugs

Question 232

Give some substrate types for P glycoprotein/MDR1

Answer 232

-Anti cancer drugs -Tyrosine kinase inhibitors -Steroids -Pesticides -Analgesic -Antibiotics -Antihistamines

Question 232

Name some substrates that P glycoprotein/MDR1 and CYP3A4

Answer 232

-Cyclosporine -Tacrolimus -Paclitaxel -Doxorubicin -Midazolam

Question 233

Describe how P53 and P glycoprotein/MDR1 interact in multidrug resistance in cancer?

Answer 233

-Wild type p53 represses MDR1 via direct DNA binding -Wild type p53 mediated down regulation of MDR1 via miR34a and LRPPRC -Mutant p53 cooperates with ETS1 to up regulate ABCB1 expression -Increasing MDR1 in cells

Question 234

Describe an alternative method to efflux for multidrug resistance via P glycoprotein/MDR1

Answer 234

-Lysosomal accumulation of anticancer drugs -Plasma membrane containing MDR1 buds inwards to form early endoscopes -As the endosome matures into a lysosome, it becomes increasing acidified -Drugs enter the cell and lysosome -Becomes charged under the acidified conditions and trapped in the lysosome and unable to reach the nucleus

Question 235

Describe BSEP transporters

Answer 235

-Coded by ABCB11 gene -"Sister of P glycoprotein" -Transports bile salts so named Bile Salt Export Pump -Expressed only in hepatocytes -Transports some drugs across canalicular membrane -Aids in multidrug resistance

Question 236

Describe MDR3 transporters

Answer 236

-Coded by ABCB4 gene -Specific translocate (floppase) for phosphatidylcholine -Into the lumen from the canalicular membrane in hepatocytes -Genetic. mutations lead to hepatobiliary diseases, affecting anthracycline, vinca alkalid and taxanes.

Question 237

Describe MRP transporters

Answer 237

-Multirdrug resistance associated protein -Encoded by ABCC1 -Range of functions from protection from xenobiotics to channeling ions -Facilitate the extrusion of glutathione, glucuronate and sulphate conjugates

Question 238

Where are MRP transporters found in the body?

Answer 238

-Barriers (Blood placenta barrier, BBB, Blood testis barrier) -Excretory and secretory (Liver, Kidney, Lungs Intestine) -Other tissues (Prostate, Muscle, Blood)

Question 239

Describe the structure of MRP1

Answer 239

-Contains 3 transmembrane membrane (TMD0, TMD1, TMD2) -TMD0 and TMB1 are attached, then to NBD1, to TMD2, then NBD2

Question 240

How doe MRP1 and MDR structures differ?

Answer 240

MRP1 contains an extra transmembrane domain, TMD0, making it heavier

Question 241

What molecules do MRP1 prefer?

Answer 241

Amphiphillic organic ions, eg LTC4

Question 242

What is LTC4?

Answer 242

-Leukotriene C4 -High affinity substrate for MRP1 -Involved in inflammation (important for asthma and allergy)

Question 243

How is LTC4 formed?

Answer 243

-By conjugation of GSH to LTA4 via catalysis by leukotriene synthase enzyme -This enzyme is active in eosinophils, monocytes, neutrophils and macrophages

Question 244

Name some Phase II conjugation reactions in the liver?

Answer 244

-Conjugation with glutathione -Conjugation with amino acids -Methylation -Glucuronidation -Acetylation -Sulfation

Question 245

What phase II products can MRP1 transport?

Answer 245

Glutathione conjugates (also uses GSH as a cotransporter)

Question 246

Name some MRP1 xenobiotic substrates

Answer 246

-Alkylating agent -Antibiotics -Antifolate -Anthracycline -Vinca alkaloids

Question 247

Name some MRP1 endobiotic substrates

Answer 247

-GSH conjugates -Folates -Sulfate conjugates -Peptides

Question 248

What have Lorico et al demonstrated with MRP1?

Answer 248

-Studied MRP1 knockout mice -No difference in viability or fertility -Elevated tissue levels of glutathione (GSH) -Unchanged tissue levels of glutathione in organs expressing no MRP1 -Increases sensitivity to several chemotherapies

Question 249

What have Johnson et al demonstrated with MDR1 and MRP1?

Answer 249

-Knockout mass have hypersensitivity to chemotherapies -However, when only one is knocked out, the other may compensate

Question 250

What regulates MRP1 expression?

Answer 250

MYCN (Blanc et al 2003)

Question 251

What did Ferrandis et al 1994 find with MYCN?

Answer 251

MYCN amplification and MDR1 over expression are frequently observed

Question 252

What is MRP2 a transporter of?

Answer 252

Glucuronides

Question 253

Where are MRP2 transporters found?

Answer 253

-Highly expressed on bile canaliculus on hepatocyte -Also on apical membranes of kidney and intestine

Question 254

What is the role of MRP2?

Answer 254

-Transport glucuronides -Eliminate drug glucuronides in bile (eg morphine) -Eliminates bilirubin from body

Question 255

How is bilirubin removed from the body?

Answer 255

MRP2 transports bilirubin glucuronide from the liver to the bile

Question 256

What is found in patients with Dubin-Johnson syndrome?

Answer 256

-No active MRP2 due to mutations -Have high levels of bilirubin glucuronide -May cause jaundice in pregnancy or with some drugs

Question 257

Describe SUR1

Answer 257

-Encoded by ABCC8 -No transporter role but acts as ATP sensitive regulator of potassium channel -Important in control of blood glucose by pancreas

Question 258

Describe how SUR1 functions?

Answer 258

-Sulfonylureas bind to receptors causing effect on K+ channel, membrane potential becomes more positive opening voltage gated Ca2+ channels -Rise in intracellular calcium leads to insulin secretion

Question 259

Name some members of the ABCB family of ABC transporters

Answer 259

-MDR1 -MDR3 -BSEP

Question 260

Name some members of the ABCC family of ABC transporters

Answer 260

-MRP1 -MRP2 -CFTR -SUR1

Question 261

Name some members of the ABCG family of ABC transporters

Answer 261

BCRP

Question 262

Describe BCRP

Answer 262

-Breast Cancer Related Protein -Encoded by ABCG2 gene -Also known as MXR -Also expressed in other tissues and relevant to drug excretion

Question 263

Describe the structure of BCRP/MXR

Answer 263

-Single spanning transporter -Contains one transmembrane domain and one NBD -Forms homodimers (BCRP) or heterodimers (ABCG5, ABCG8)

Question 264

Where is BCRP located primarily?

Answer 264

-Lactating breasts -Also located in liver, kidney, placenta, testis, colon

Question 265

What is the implication of BCRP's location on healthcare?

Answer 265

-Secretion of xenobiotics into milk -Meaning restriction on use of certain drugs by nursing mothers

Question 266

What is Imatinib a substrate for?

Answer 266

BCRP/ABCG2

Question 267

Describe how Chronic myeloid leukaemia (CML) cells become resistant to Imatinib?

Answer 267

-CML stem cells appear to have higher levels of ABCG2 -Decreased levels of regulatory miRNA-212 results in increased ABCG2 levels -ABCG2 transports imatinib

Question 268

Describe how imatinib can help treat cancer

Answer 268

-Inhibits BCR ABL (constitutively active tyrosine kinase driving cell division and inhibiting apoptosis) -Leads to down regulation of BCRP

Question 269

How are ABCG2/BCRP and gout linked?

Answer 269

-GWAS showed a genetic polymorphism resulting in an amino acid change in BCRP is risk factor -Uric acid (which accumulates in joints) is a substrate for BCRP, which unstable forms of these proteins are unable to excrete this.

Question 269

Describe Tyrosine Kinase inhibitors

Answer 269

-Target specific signalling pathways deregulated in cancers -Targeted treatment for cancers -Non toxic and more specific -There are risks of acquired resistance

Question 270

What is the mutation in ABCG2/BCRP that leads to gout?

Answer 270

-Q141K -Causes change in NBD structure

Question 271

How do ABC transporters affect drug development?

Answer 271

-Drugs must be tested for ABCs as substrates -As this may effect the ability of treatments

Question 272

Name some strategies to overcome ABC mediated Multidrug resistance?

Answer 272

-Novel delivery systems -Agents that bypass transporters -Reduce expression of ABCs eg siRNA, miRNA -Antibodies -Natural compounds -Chemical Inhibitors

Question 273

Describe and name MDR1 inhibitors

Answer 273

-Inhibitors of MDR1 to overcome multidrug resistance in cancer -Verapamil (first gen), Valspodar (second gen), Dofequidar (third gen)

Question 274

What is an added benefit of Verapamil (MDR1 inhibitor)?

Answer 274

It sensitises MDR cells to chemotherapy

Question 275

Describe collateral sensitivity in the context of MDR1 inhibitors?

Answer 275

-Cancer cells overexpress MDR1/Pglycoprotein, and become hypersensitive to certain compounds -This stimulates GSH efflux -This offers a strategy to target drug resistant cancer cells ONLY -As drugs accumulate in these cells and not others

Question 276

What drugs do 5681014 and BSO sensitise MRP1 expressing cells to?

Answer 276

Chemotherapies such as vincristine and doxorubicin

Question 277

Name some inhibitors of ABCG2/BCRP

Answer 277

-Verapamil -Ritonavir -Elacridar -Novobiocin

Question 278

What are the issues surrounding targeting ABC transporters as a treatment for cancer?

Answer 278

-Physiological roles of ABC transporters -Ubiquitious expression -Transporter redundancy -Dose adjustment and monitering when combining with drugs with narrow therapeutic window

Question 279

What aspects of cancer do ABC transporters lead to?

Answer 279

-Inflammation -Cancer stem like cells -Anti apoptosis -Sustained proliferation -Metastasis and invasion

Question 280

Why is cell signalling important?

Answer 280

To transfer information -From environment to cell -From cell to cell To enable homeostasis by reacting accordingly to changes

Question 281

When cell signalling fails, what may occur?

Answer 281

Pathology

Question 282

What aspects of physiology may cell signalling affect?

Answer 282

-Metabolism -Nervous system -Physiology -Pharmacology -Cell cycle -Immunology -Development

Question 283

What are most signals that a cell responds to?

Answer 283

-Most are chemical (eg hormones, neurotransmitters, pheromones) -Some are physical (eg light, heat, pressure)

Question 284

What are the basic features of cell communication

Answer 284

-Synthesis of chemical signal -Secretion/Release -Reception (cells decode this interaction) -Response

Question 285

Give some methods of cell to cell communication

Answer 285

-Gap junction -Paracrine and autocrine signalling -Hormonal -Neurohormonal -Neurotransmitter

Question 286

Give some important cell signalling pathways

Answer 286

-Steroid hormone -Ligand gated ion channels -Cyclic AMP pathway -Phosphoinositide pathway -Tyrosine kinase pathway

Question 287

Give the steps in a cell signal processing pathway and examples of components

Answer 287

-Chemical signal (hormone) -Receptor (ion channel linked) -Transducer (G protein) -Amplifier (Adenylyl cyclase) -Secondary messenger (cAMP) -Effectors (Protein kinases) -Response element (enzyme) -Response (Metabolism)

Question 288

What are the basic principles we find in cell signalling processing

Answer 288

-Amplification -Heterogeneity (each pathway component often has multiple forms) -Information transfer -Dynamics (responses are dependent on both temporal and spatial aspects)

Question 289

Describe phosphorylation as a method of covalent modification

Answer 289

-The addition of a terminal phosphate group of ATP to hydroxyl group of specific amino acid within a target protein (most commonly serine and threonine) -By a protein kinase, acting on residues lying within a specific consensus motif -Changing activity or function of the protein

Question 290

What is the difference in types of phosphorylation kinase enzymes?

Answer 290

Type II PKA forms a stable interaction with AKAPs, via the R subunits, so it is not free in the cytosol (type I is). When cAMP binds to the R subunits of type II the catalytic subunits are not released (type I is).

Question 291

How many temporal changes may be different in cell signalling?

Answer 291

Differences in frequency or duration

Question 292

What is luminescence?

Answer 292

Emission of light by a substance not resulting from heat

Question 293

Give some sources of bioluminescence?

Answer 293

-Luciferase (firefly tails) -Aequorin (Jellyfish)

Question 294

What is phosphorescence?

Answer 294

-Light emission continuing for a longer time after the excitation source is removed due to energy being trapped in metastable state

Question 295

What is fluorescence?

Answer 295

Emission of light by a substance that has absorbed light

Question 296

What is autofluorescence?

Answer 296

-Natural emission of light by biological structures when they absorb light (UV or visible) -Caused by intrinsic fluorophores (eg NADH, Porphyrins, Elastin)

Question 297

Describe the energy changes in fluorescence?

Answer 297

-When light is put in, ground state is energised to excited state -Excited state then transfers to ground state, releasing energy (light)

Question 298

What is fluorescein?

Answer 298

-Synthetic compound that exhibits strong fluorescence -Used in ophthalmology, immunofluorescence and as a tracer in physiology

Question 299

What is the dichroic filter a component of?

Answer 299

Widefield fluorescence microscope

Question 300

What are the three critical filters needed for a fluorescence microscope?

Answer 300

-Excitation -Dichroic -Emission

Question 301

What is the function of the dichroic filter block?

Answer 301

Excitation filter - Selects the appropriate wavelength of light to excite the fluorophlore Dichroic mirror - Reflects excitation light toward the sample and transmits emitted fluorescence toward the detector Emission filter - Allows only the fluorescence emission light to reach the detector

Question 302

Describe the light path through a dichroic filter block

Answer 302

-Light from the source is passed through the excitation filter, isolating the excitation wavelength -Filtered light is reflected by the dichroic mirror onto the sample -Fluorescent light emitted is collected and transmitted back through the dichroic mirror and then passed through the emission filter to the detector

Question 303

What is the benefit of confocal fluorescence microscopy over widefield fluorescence

Answer 303

Produces higher resolution and contrast images, eliminating background noise and out of focus light

Question 304

How do you calculate resolution?

Answer 304

Lateral resolving power = Wavelength of light / (2 x Numerical aperture of lens)

Question 305

What are the principles of stimulated emission depletion imaging?

Answer 305

-Surpasses the diffraction limit of conventional light microscopy -By combining laser beams and precise optical control to restrict fluorescence emission to a sub diffraction sized region -Enhancing resolution to as fine as 20-50nm (far below 200nm limit)

Question 306

Give some methods to fluorescence label microscopy samples

Answer 306

-Small molecule probes eg -dye chemistry -antibodies -Create fluorescent proteins

Question 307

What are the issues with preparing fluorescent labels in microscopy?

Answer 307

-Getting probe to target -Only labelling the target -Overcoming any sample autofluorescence -Phototoxicity -Photobleaching

Question 308

Give some of the issues with using small molecular probes in fluorescence microscopy

Answer 308

-Dye chemistry -Many cannot be fixed -Few target individual proteins -Immunofluorescent techniques -Difficult to use with live cells -Only cell surface proteins are visible -Getting label to target requires permeabilisation

Question 309

What are some of the issues surrounding using fluorescent proteins in microscopy?

Answer 309

-Not native proteins -Strong promoters can enhance signal -May perturb protein function

Question 310

Give some fluorescence microscopy applications

Answer 310

-Live organelle/protein tracking -Measuring interaction/association via colocalisation -Identify cells that coexpress certain proteins -Visualise molecular association (via FRET)

Question 311

What is the function of FRET in fluorescence microscopy

Answer 311

-Forster Resonance Energy Transfer -Established to measure the interaction and location of interaction of two proteins or structures -Can measure very close interactions (1-10nm)

Question 312

Describe how FRET works?

Answer 312

-Donor fluorophore is excited by an external light source -If the acceptor fluorphore is within the FRET distance of the donor and their emission and excitation spectra overlap, the excited donor transfers its energy to the acceptor without emitting a photon -The acceptor fluorophore emits light at its characteristic emission wavelength after being excited by the energy transfer

Question 313

Which fluorophores are used in FRET?

Answer 313

CFP (cyan fluorescent protein) as the donor and YFP (Yellow fluorescent protein) as the acceptor

Question 314

What can FRET be used to study?

Answer 314

cAMP signalling

Question 315

What are neurotransmitters?

Answer 315

Chemical messengers, released from one neurone (presynaptic cell) acting at a close site on another (postsynaptic cell) to elicit an effect determined by the specific nature of the receptor

Question 316

Give some key neurotransmitters

Answer 316

-Acetylcholine (voluntary movement of the muscles) -Glutamate (excitatory neurotransmitter) -Dopamine (Motivation, pleasure) -Serotonin (emotions, wakefulness and temp reg) -GABA (inhibitory neurotransmitter) -ATP (neuronal/glial communication)

Question 317

Describe ionotropic channels

Answer 317

-Channel opens when neurotransmitter binds to the receptor -Allowing ions to flow directly across the membrane -Causing immediate changes

Question 318

Describe metabotropic channels

Answer 318

-Ligand binding activates intracellular signalling pathways -Usually involving G proteins and second messengers -May modulate ion channels indirectly

Question 319

What are reversal potentials?

Answer 319

-Membrane potentials at which there is no net flow of ions through an ion channel -Driving forces of the electrochemical gradient are perfectly balanced -ie Concentration gradient = Electrical gradient

Question 320

What is a quanta?

Answer 320

A quantum represents the neurotransmitter content of a single synaptic vesicle, which is released into the synaptic cleft

Question 321

When synaptic vesicles spontaneously release neurotransmitters, what are produced?

Answer 321

Miniature end plate potentials (MEPPs)

Question 322

Describe Nicotinic acetylcholine receptors

Answer 322

-Found at the neuromuscular junction (muscle cells) and between pre and post synaptic cells in the ANS -Permeable to Na+, K+ and Ca2+ -Erev of -5mV

Question 323

What do nicotinic acetylcholine receptors consist of?

Answer 323

5 subunits - 2⍺, 1β, 1𝛅, 1𝛆

Question 324

What subunit does acetylcholine bind to on nicotinic acetylcholine receptors?

Answer 324

⍺ subunit, meaning 2 can bind per receptor

Question 325

Name some antagonists for nicotinic acetylcholine receptors

Answer 325

⍺-bungarotoxin (snake venom) and curare compounds (plants)

Question 326

Describe what causes Myasthenia Gravis?

Answer 326

-Autoimmune disease -Body produces autoantibodies against nicotinic ACh receptors -End plate potential can't generate muscle stimulation

Question 327

What type of receptors are those for GABAa, GABAc, and Glycine?

Answer 327

Ionotropic

Question 328

What type of receptors are those for GABAb?

Answer 328

Metabotropic

Question 329

What are GABA and Glycine receptor channels permeable to?

Answer 329

Cl- and HCO3-

Question 330

What links GABAa, GABAc and Glycine in structure?

Answer 330

All pentameric structures

Question 331

What do the subunit composition of GABA receptors dictate?

Answer 331

-Receptor properties (sensitivity, conductance, kinetics of opening and closing) -Cell surface distribution -Dynamic regulation

Question 332

Describe 𝛿 subunits of GABA receptors

Answer 332

-Sensitive with low desensitisation -Mediates tonic GABAergic currents

Question 333

Describe how benzodiazepines modulate GABAa receptors?

Answer 333

-Mediate sedation -Bind at interface of ⍺/γ2 subunits -Eases opening of receptors

Question 334

Describe how barbituates modulate GABA?

Answer 334

-Increase the affinity of GABA -Locks GABA receptors in the open state

Question 335

Describe how alcohol modulate GABA?

Answer 335

Enhances GABA action

Question 336

What are the agonists for purinergic receptors?

Answer 336

P1 - Adenosine P2Y- ATP (and other nucleotide triphosphate) P2X ATP

Question 337

What are the functions of purinergic receptors?

Answer 337

-Widespread glia and neuronal expression -ATP released in synaptic vesicles -An important means of neuronal to glial, and glia-to-glia communication

Question 338

Describe the role of ionotropic glutamate receptors

Answer 338

-Transient opening of ion channels allowing net influx of cations -Generating an excitatory current

Question 339

Describe the role of metabotropic glutamate receptors

Answer 339

-Modulatory role in synaptic transmission -Roles in learning, memory, and pathology (epilepsy, schizophrenia)

Question 340

Name some classes of ionotropic glutamate receptors

Answer 340

-AMPA -NMDA -Kainate

Question 341

Name which classes of ionotropic receptors Glutamate and Kainate activate?

Answer 341

All! :D AMPA, NMDA, and Kainate

Question 342

Name which classes of ionotropic receptors Kainate activate?

Answer 342

Kainate agonist has no effect on AMPA or NMDA

Question 343

Where are ionotropic glutamate receptors localised?

Answer 343

-Usually colocalised at synapses -Can be both synaptic and extrasynaptic -Can be both pre synaptic (autoreceptors) and post synaptic

Question 344

What cations do ionotropic glutamate receptors transport?

Answer 344

-Ca2+ -Na+ -K+

Question 345

Do ionotropic glutamate receptors work alone or together?

Answer 345

They do not work alone, and are part of an enormous and complex protein network

Question 346

Describe the structure of ionotropic glutamate receptors

Answer 346

-Comprised of 4 subunits (tetramer) -Three transmembrane domains (TM1, TM3 and TM4) and a reentrant loop (TM2)

Question 347

What are the two types of modifications that AMPAR subunits can undergo to influence receptor kinetics?

Answer 347

-Alternative splicing (flip or flop, long or short tail) -RNA editing (Q/R, R/G)

Question 348

What are the subunits for AMPA receptors called?

Answer 348

GluA1 - GluA4

Question 349

In heteromeric (subunit) AMPA receptor channels, what does the presence of edited GluA2 determine?

Answer 349

-The current voltage curve -Ca2+ permeability

Question 350

What can current flow through AMPA receptors be terminated by?

Answer 350

-Deactivation (agonist unbinding leading to closure of channel, requiring removal of transmitter) -Desensitisation (the channel closes whilst agonist remains bound)

Question 351

What do flip/flop variants of AMPA receptors affect?

Answer 351

-Receptor kinetics -ie the speed at which the receptor desensitises and closes

Question 352

What does editing of the Q/R site on AMPA receptors affect?

Answer 352

-Ca2+ permeability -Acting on GluA2 receptors (swapping glutamine for arginine, which renders GluA2 impermeable)

Question 352

Describe Kainate receptors

Answer 352

-Ionotropic glutamate receptors -Involved in synaptic transmission -Contributes to plasticity and long term potentiation -Potent agonist is kainate and glutamate

Question 353

Describe NMDA receptors

Answer 353

-Ionotropic glutamate receptors -Involved in synaptic transmission, plasticity and excitatory signalling -Binds agonist glutamate and coagonist glycine

Question 354

Describe what activates NMDA receptors

Answer 354

-Blocked by Mg2+ "cork" -Upon depolarisation, this cork "uncorks" -Allowing free movement of Ca2+ and Na+ through the channel

Question 355

Give an example of evidence that NMDA is involved in memory and learning

Answer 355

-Rats are treated with NMDA agonist AP5 -Found to impair spatial memory

Question 356

What is spike timing dependent synaptic plasticity

Answer 356

-Synaptic plasticity in which the timing of action potentials determines whether the strength of a synapse is increased (LTP) or decreased (long term depression) -Caused by NMDA receptors (higher calcium influx through triggers LTP, lower triggers LTD)

Question 357

How does calcium lead to specific cellular responses?

Answer 357

-Compartmentalisation of Ca2+ entry -Localisation of Ca2+ binding proteins

Question 358

What is the phosphoinositide pathway an example of?

Answer 358

Second messenger model pathway

Question 359

How is intracellular calcium concentration kept low?

Answer 359

Through OFF mechanisms

Question 360

Give some cellular responses to rises in intracellular calcium concentration

Answer 360

-Changes in ion permeability -Secretion -Contraction -Metabolism -Fertilisation -DNA synthesis -Development

Question 361

Name some OFF mechanisms for maintaining low intracellular calcium concentrations

Answer 361

-Ca ATPase pumps (SERCA and PMCA) -Na+/Ca2+ exchanger -Mitochondria and other organelles -Proteins (buffers and sensors)

Question 362

Name some Calcium ATPase pumps

Answer 362

-PMCA (Plasma membrane Calcium ATPase) -SERCA (SarcoEndoplasmic Reticulum Calcium ATPase)

Question 363

Name a key protein buffer against intracellular calcium

Answer 363

Parvalbumin

Question 364

Name a key protein sensor for intracellular calcium

Answer 364

Calmodulin

Question 365

Why are high levels of intracellular calcium cytotoxic?

Answer 365

Calcium binds with phosphates to create the very insoluble aggregate calcium phosphate (what bones are made of)

Question 366

At what plasma calcium concentrations do OFF mechanisms work at?

Answer 366

Varying, however from lowest to highest: -Endoplasmic reticulum -PMCA -Na+/Ca2+ exchanger -Mitochondria

Question 367

Give the intracellular and extracellular concentrations of calcium

Answer 367

Intracellular = ~100nM Extracellular = 2-3mM

Question 368

Name some second messengers in the phosphoinositide pathway, and what they act on

Answer 368

-IP3 acts on IP3 Receptors -cADPR acts on Ryanodine receptors

Question 369

What is the effect of IP3 binding to its receptors?

Answer 369

Opens channels in ER or SR, allowing Ca2+ influx into cytosol

Question 370

What is the effect of Ryanodine receptor binding?

Answer 370

Opens channels in ER or SR, allowing Ca2+ influx into cytosol

Question 371

What are the three types of Ca2+ signal?

Answer 371

-Elementary events -Intracellular global Ca2+ wave -Intercellular global Ca2+ wave

Question 372

Describe the phosphoinositide pathway up to the production of IP3 and DAG

Answer 372

1 - Chemical signal induces a confirmational change in a 7 membrane spanning receptor 2 - The III cytoplasmic loop of the receptor activates a G protein (Gq) 3 - Gq stimulates amplifying enzyme Phospholipase C 4 - PLC acts on membrane phospholipid PIP2 to give Diacylglycerol (DAG) and Inositol 1,4,5-trisphosphate (IP3)

Question 373

Describe the phosphoinositide pathway following the production of IP3 and DAG

Answer 373

1 - DAG acts within the lipid bilayer to stimulate Protein Kinase C, IP3 diffuses into the cytosol where it acts on IP3 receptors (stimulating Ca2+ release) 2 - Onset of the calcium signal is augmented by Calcium induced calcium release (CICR) 3 - Ca2+ can act through a protein kinase or use specific binding proteins (eg calmodulin) or act directly on ion channels

Question 374

What does Phospholipase C-β act on?

Answer 374

Acts on PIP2 to produce diacylglycerol (DAG) and Inositol 1,4,5-trisphosphate (IP3)

Question 375

Describe what the calcium signal takes the form of?

Answer 375

A series of Ca2+ spikes, with increasing stimulus leading to increasing frequency

Question 376

Describe Calcium Induced Calcium Release

Answer 376

-Ca2+ ion spike feed forward to promote Ca2+ release from sarcoplasmic/endoplasmic reticulum -Causing a calcium wave through the cell -What is known as a "regenerative" Ca2+ release

Question 377

Describe the components of intracellular calcium signals?

Answer 377

A temporal aspect (the spike) and a spatial aspect (the wave)

Question 378

What is the calcium wave a correlate of?

Answer 378

The spatial correlate of the spike

Question 379

What is the calcium spike a correlate of?

Answer 379

The temporal correlate of the wave

Question 380

Without calcium induced calcium release, what would occur?

Answer 380

-Would rely upon diffusion, which is slow and ineffective due to buffering proteins before sensing proteins are stimulated -Leading to no wave or spike -CICR helps to saturate buffers

Question 381

Describe the differing distribution of IP3 receptors and Ryanodine receptors

Answer 381

-Hepatocytes contain only IP3Rs -Cardiac myocytes contain mostly RyRs -Some cells (eg neurones, secretory cells, eggs) contain both IP3Rs and RyRs

Question 382

Which receptor propagates CICR and calcium waves?

Answer 382

Both IP3 receptors and Ryanodine receptors

Question 383

What coagonists allow the release of Calcium from SR or ER

Answer 383

Agonist = Calcium Coagonist = IP3 or cADPR

Question 384

Describe the biphasic regulation of cytosolic Ca2+

Answer 384

-Low Ca2+ leads to little Ca2+ release (via CICR) but high Ca2+ concentration inhibits calcium ion release

Question 385

Describe the differing types of inhibition of IP3 receptors and Ryanodine receptors

Answer 385

IP3 receptors are truly inhibited at high [Ca2+], but RyRs plateau at high [Ca2+] (ie not complete inhibition)

Question 386

What is the action of cADPR on RyRs?

Answer 386

Theorised to sensitise RyRs to CICR

Question 387

What do both IP3 receptors and Ryanodine receptors require for functioning?

Answer 387

-Calcium binding -ATP binding (but not hydrolysis)

Question 388

Why do both IP3 receptors and Ryanodine receptors require ATP binding

Answer 388

Thought to help regulate, as sick or diseased cells (with low ATP) can not generate Ca2+ signals as they wouldn't be able to use OFF mechanisms to remove Ca2+

Question 389

Describe Elementary calcium events

Answer 389

-Do not propagate to waves -Limited to certain areas of the cell -Have physiological events -Around 5 microns in diameter -Around 200ms in duration

Question 390

Describe elementary calcium events acting on IP3 receptors

Answer 390

-Calcium Puff -Acting on about 10 receptors

Question 391

Describe elementary calcium events acting on Ryanodine receptors

Answer 391

-Calcium Spark -Acting on about 10 receptors

Question 392

What are elementary calcium release events caused by?

Answer 392

Intermediate amounts of chemical stimulus

Question 393

What are smaller than elementary calcium release events?

Answer 393

Fundamental events called blips (IP3) and quarks (RyR)

Question 394

Give an example of how elementary calcium release events having a physiological effect

Answer 394

-If calcium sparks (in smooth muscle cells) are close enough to K+ channels (activated by Ca2+) -Leads to opening and hyperpolarisation, leading to muscle relaxation

Question 395

What are intercellular Ca2+ waves propagated using?

Answer 395

-Gap junctions -IP3 AND Ca2+ needs to move through the junction -Can propagate between different cell types

Question 396

Describe the sequence in the calcium signalling pathway that leads to the breakdown of glycogen into glucose in liver cells?

Answer 396

-Calcium binds to Calmodulin sensing protein -Ca-calmodulin binds to Kinase II, which phosphorylates phosphorylase kinase -This phosphorylates phosphorylase -Which converts glycogen into glucose

Question 397

How does higher frequency of calcium spikes increase Kinase II activation?

Answer 397

At higher frequencies, more time is spent in its activated state (not deactivating as it doesn't have enough time)

Question 398

What can influence the output of the phosphoinositide pathways?

Answer 398

-Different isoforms -eg Type I, II and III IP3 receptors -eg Type I, II, and III RyRs

Question 399

How may cells differentiate in their phosphoinositide pathways?

Answer 399

During development, a process of signalsome expression results in the appearance of cell type-specific signalsomes to create the normal output signals used to control particular cellular functions

Question 400

Give the methods to which signalsomes can be remodelled

Answer 400

-Phenotypic remodelling (eg phosphorylation, altering transcription rate of component) -Genotypic remodelling (eg somatic or gremlin mutations alters activity of component)

Question 401

Describe how phenotypic remodelling of the calcium signalsome can increase force of contraction in the heart

Answer 401

cAMP dependent reversible phosphorylation of key Ca2+ signalling components (eg VOC, SERCA) enables heart cells to generate larger Ca2+ signals

Question 402

Describe how phenotypic remodelling of the calcium signalsome can aid liver cell regeneration

Answer 402

-Down regulation of key Ca2+ signalling components results in lower frequency Ca2+ spikes of greater duration

Question 403

Describe phenotypic remodelling of the calcium signalsome resulting in Alzheimer's disease

Answer 403

-Extracellular plaque deposits of the β amyloid peptide, disrupting synaptic transmission -β amyloid oligomers increasing Ca2+ entry via the NMDA receptor -The amyloid precursor protein intracellular domain (AICD) increasing Ca2+ release from stores -This abnormal amyloid metabolism results in an up regulation of neuronal Ca2+ signalling to induce an initial decline in memory

Question 404

How does amyloid precursor protein domain (AICD) increase calcium within the cell?

Answer 404

-Increases Ca2+ leak through RyR -Decreases calcium buffering by calbindin

Question 405

Describe the differing calcium levels between in healthy vs Alzheimer's patients

Answer 405

Healthy - Calcium remains at 100nM until spikes with long term potentiation. Raises to 300nm in sleep to erase temporary memories during sleep. Alzheimer's - Calcium remains at 300nm continuously, meaning any temporary spikes and memories formed are erased instantaneously.

Question 406

Describe how positive feedback is critical for Alzheimer's disease progression

Answer 406

-Bidirectional relationship between Ca2+ signalling and the amyloidogenic pathway -While the amyloids stimulate an increase in Ca2+, the latter can stimulate the metabolism of APP -Producing an element of positive feedback

Question 406

Describe how Calcium signalsome genotypic remodelling can result in Brody myopathy

Answer 406

-Skeletal muscle genetic disorder characterised by stiffness and cramp brought on by prolonged Ca2+ elevation and a slowing of relaxation -Defect results from mutation in SERCA1 pump -SR unable to refill with Ca2+; cytosolic Ca2+ remains elevated so relaxation impeded

Question 407

Give some examples of Calcium signalsome remodelling in cancerous cells

Answer 407

-Altered SERCA pump activity -Altered Ca2+ release through IP3 receptors -Altered resting level of Ca2+

Question 408

Describe the sequence in the cAMP signalling pathway?

Answer 408

GPCR->Adenylyl cyclase->cAMP->PKA->Cellular response

Question 409

What are the two major types of Adenylyl cyclase

Answer 409

-Membrane bound Adenylyl Cyclase -Soluble Adenylyl Cyclase

Question 410

Describe Membrane bound adenylyl cyclase

Answer 410

-Integral in plasma membrane -9 Isoforms (AC 1-9) -Convert ATP to Cyclic AMP -Regulated by GPCRs, calcium/calmodulin, protein kinases or Forskolin

Question 411

What is Forskolin?

Answer 411

A plant-derived diterpene (alkaloid) that directly activates most AC isoforms by binding to their catalytic domains.

Question 412

What GPCRs regulate membrane bound adenylyl cyclase?

Answer 412

-Activated by Gαs -Inhibited by Gαi

Question 413

Name some off mechanisms in the cAMP signalling pathway

Answer 413

-Inhibit cAMP production -Breakdown cAMP -Remove cAMP from cell

Question 413

Describe soluble Adenylyl cyclase

Answer 413

-Found in cytoplasm, unassociated with membrane -Regulated by bicarbonate ions (activating, and calcium -Involved in metabolic regulation, sperm motility, intracellular pH sensing

Question 414

Name a membrane bound adenylyl cyclase that is regulated by cytosolic calcium

Answer 414

Adenylyl Cyclase 8

Question 415

Describe cAMP production inhibition

Answer 415

Some GPCR agonists activate the inhibitory G-protein, Gαi which reduces adenylyl cyclase activity, opposing stimulation by Gαs and thereby lowering cAMP levels

Question 416

Describe the breakdown of cAMP

Answer 416

-By phosphodiesterases (PDEs) -Expression of isoform is tissue specific -Leading to "shaping" the local cAMP signal's duration, amplitude, and spatial localisation

Question 417

How many isoforms of phosphodiesterases exist?

Answer 417

-11 Isoforms (PDE 1-11) -8 Breakdown cAMP whilst others breakdown cGMP

Question 418

What inhibits PDEs?

Answer 418

-Caffeine (increasing cAMP level) -Cilastazol (causes vasodilation increasing peripheral vascular flow) -Milrinone (Increases heart rate and isotropy to fight failing hearts) -Roflumilast (relaxes airway smooth muscle to reduce airway obstruction)

Question 419

Name what removes cAMP from the cell, and an example.

Answer 419

-Range of plasma membrane ABC transporters -e.g MRP4

Question 420

What are some issues with the "linear" model of cAMP signalling, and what do these issues indicate?

Answer 420

-Different agonists increase cAMP levels but produce distinct responses in the same cells -Some 'physiological' agonists produce cAMP dependent responses but do not appear to change global cAMP levels INDICATING that changes in cAMP must be highly localised to spatially distinct areas inside cells (called microdomains) and changes in cAMP are likely to be agonist specific

Question 421

How is cAMP signalling compartmentalised?

Answer 421

-GPCRs localised to different regions of the cell -Restrict diffusion of cAMP from the plasma membrane to cytosol (by phosphodiesterases) -Target PKA to distinct sites and substrates in cells (using AKAPs)

Question 422

What are AKAPs?

Answer 422

A-Kinase Anchoring Proteins (AKAPs)

Question 423

Describe what AKAPs are, and name key aspects of their structure

Answer 423

-Scaffolding proteins, regulating spatial and temporal aspects of cellular signalling pathways -Contains: -PKA binding (docking) domain -Targeting domain -Multiprotein complex formation regions

Question 423

Describe how AKAPs work?

Answer 423

-PKA binding domain anchors the PKA holoenzyme to specific locations within the cell (positioning it near its substrates) -Targeting domain directs the AKAP to certain cellular compartments eg plasma membrane, nucleus, etc -AKAPs assemble signalling complexes to form signalsomes

Question 424

Which PKAs do AKAPs bind to?

Answer 424

PKA type II

Question 425

Which AKAP is involved in CFTR activation by PKA?

Answer 425

Ezrin targets PKA to CFTR, requiring an adapter protein (NHERF1)

Question 426

Describe NHERF1

Answer 426

-Helps Ezrin target PKA to CFTR -Contains: -PDZ1 binding domain that CFTR binds to -ERM domain that binds Ezrin

Question 427

Describe the two approaches to measuring cAMP dynamics in living cells

Answer 427

-FRET, using a cAMP BD which has two different fluorescent proteins (CFP and YFP) attached that undergo FRET, with FRET decreasing when cAMP rises -Intensity, cAMP BD + fluorescent protein tag, rise in cAMP increases fluorescent intensity

Question 428

Describe the mechanisms of phenotypic remodelling in the cAMP signalsome

Answer 428

-Changes in component expression (Upregulation, Downregulation or alternative splicing) -Alterations in localisation (of separate components of the signalsome) -Dynamic assembly and disassembly -Post translational modifications (eg phosphorylation) -Receptor desensitisation and cross-talk (can integrate multiple signals from other pathways eg calcium)

Question 428

Describe phenotypic remodelling of the cAMP signalsome in cardiac cells

Answer 428

-Activation of β-adrenergic receptor elevated cAMP localised to T-tubules and the SR -Phosphorylation of key proteins involved in excitation-contraction coupling, including LTCCs, Phospholamban and Ryanodine 2 receptors -Activation of prostanoid receptors led to phosphorylation of enzymes involved in metabolism as well as transcriptional factors involved in long-term cellular changes. -Inhibiting PDEs, abolished receptor-dependent cAMP signalling as well as selective PKA phosphorylation.

Question 429

Describe how phenotypic remodelling by the cAMP pathway interacts with calcium signalling, helping to coordinate cardiac muscle activity

Answer 429

The increased force of contraction induced by β-adrenergic stimulation during exercise, or the fight-or-flight response, leads to PKA-dependent phosphorylation of key Ca2+ signalling components: LTCCs, Phospholamban, and Ryanodine 2 receptors -Enabling heart cells to generate larger Ca2+ signals (increase inortopy), remove Ca2+ quicker into stores during diastole (lustropy) and reload Ca2+ stores better to increase inotropy

Question 430

How does PKA increase LTCC activity?

Answer 430

-PKA phosphorylation increases open state probability of individual LTCCs in the sarcolemma -PKA also recruits more LTCCs to sarcolemma

Question 431

What is the effect of SERCA phosphorylation during phenotypic remodelling by the cAMP pathway?

Answer 431

-Activates it -Increasing Ca2+ cycling -Increasing ATP consumption -Increasing chronotropy and inotropy

Question 432

Name protein kinases activated by cGMP

Answer 432

Protein Kinase G

Question 433

Name protein kinases activated by Ca2+

Answer 433

-PKC -Ca2+/Calmodulin dependent PKs

Question 434

Describe the steps in the cyclic GMP signalling pathway

Answer 434

-Guanylyl Cyclases are activated by either nitric oxide (synthesised by NOS) or extracellular ligands, converting GTP to cGMP -cGMP either activates PKG, CNG channels, or regulates PDEs -cGMP is degraded to GMP by phosphodiesterases (PDEs)

Question 435

What are the two types of guanylyl cyclases in the cGMP signalling pathway?

Answer 435

-Soluble form (sGC) activated by nitric oxide -Plasma membrane bound form (pGC) activated by a smaller number of peptide agonists

Question 436

What breaks down cGMP?

Answer 436

cGMP-dependent PDEs

Question 437

Name some ligands that may bind to plasma membrane bound guanylyl cyclases

Answer 437

-Atrial natriuretic factor (ANP) -Brain natriuretic factor (BNP) -C type natriuretic factor (CNP) -Guanylin

Question 438

Give some examples of cyclic GMP signalling in health and treatments

Answer 438

-Nitric oxide is released from endothelial cell, activating sGC/cGMP in VSM leading to vasodilation and fall in BP. NO generating drugs are often used to treat angina -Viagra, a type 5 cGMP PDE inhibitor causes rise in cGMP, relaxing smooth muscle in some tissues. Used to treat erectile dysfunction and pulmonary hypertension

Question 439

Give some examples of cyclic GMP signalling in disease

Answer 439

-Heat stable enterotoxin from E coli in intestine activates pGC/cGMP, causing PKG to phosphorylate and activate CFTR, leading to a secretory diarrhoea -LPS (endotoxin) from gram negative bacteria, increases expression in inducible NO synthase, causing excessive NO production. Leads to clinical shock due to severe drop in BP

Question 440

What are the 3 groups of protein kinase C?

Answer 440

-Conventional (require both DAG and calcium for activation) -Novel (Require DAG but are calcium independent) -Atypical (independent of DAG and calcium)

Question 440

What are the the domains that make up protein kinase C?

Answer 440

-Regulatory domains -Including pseudosubstrate region that mimics the substrate, keeping the enzyme inactive by blocking catalytic domain -Hinge region -Catalytic domain (consisting of N lobe and C lobe)

Question 441

Describe activation of PKC

Answer 441

-Ca2+ binds to C2 domain, PKC then translocates to the PM, binding to DAG via C1 domain -Interaction with cofactors displace the pseudosubstrate from C4 domain, activating the kinase -PKC undergoes autophosphorylation, stabilising its active confirmation

Question 442

What can all isoforms of PKC be activated by?

Answer 442

Phorbol esters - plant alkaloids (are tumorigenic)

Question 443

What are the two types of Ca2+ or calmodulin dependent protein kinases?

Answer 443

-Multifunctional CaM kinases (eg CaMKII) which are widespread in cells and tissues -Substrate specific CaM kinases (eg CaMKK) which are specialised in specific cells

Question 444

Give some functions of CAMKII

Answer 444

-Regulates activity of NMDA receptors by phosphorylating sites on both NR2A and NR2B (AT ligand gated ion channel lectures) -Enhances IP3 formation by inhibiting inositol polyphosphate 5-phosphotase -Central role in frequency decoding of calcium signals, as well as in Long term potentiation and Long term depression -Phosphorylates PLB to control SERCA2 pump

Question 445

Describe protein phosphatases

Answer 445

Remove phosphate groups from phosphorylated proteins (Ser/Thr and Try residues)

Question 446

Name some chemical inhibitors of protein phosphatases

Answer 446

-Okadaic acid (blocks PP1 and PP2A) -Cyclosporin A (very specific for calcineurin (PP2B), used clinically for immunosuppression via effects on T cells

Question 447

Describe Okadaic acid as a chemical inhibitor of protein phosphatases

Answer 447

-Toxin produced by algae which accumulate in marine sponges and shellfish -When ingested cause diarrhetic shellfish poisoning -Caused by -Increased paracellular permeability -More active CFTR promoting fluid loss from GI tract -INHIBITS PROTEIN PHOSPHATASES

Question 448

Describe how phosphorylase kinase requires both PKA phosphorylation and Ca2+ binding to be fully active in skeletal muscle

Answer 448

-The 𝛿 subunit is calmodulin (requires calcium to bind) -γ is the catalytic subunit -⍺ and β subunit phosphorylation are required, increasing the calcium sensitivity of phosphorylase kinase

Question 449

What blood glucose concentration is found in healthy vs diseased states (diabetes)?

Answer 449

Healthy = ~5mM Diseased = Can rise to ~60mM

Question 450

What two major hormones are involved in blood glucose homeostasis?

Answer 450

Insulin and Glucagon

Question 451

What is the effect of hyperglycaemia on the hormones involved in blood glucose homeostasis?

Answer 451

-Stimulates insulin secretion -Inhibits glucagon secretion

Question 452

What processes does insulin stimulate?

Answer 452

-Glucose uptake into muscular cells -Increased glycogenesis in liver -Decreased gluconeogenesis in liver

Question 453

What processes does glucagon stimulate?

Answer 453

-Glycogenolysis in liver -Gluconeogenesis in liver

Question 454

Describe pancreatic islets of langerhans

Answer 454

-Scattered throughout the pancreas -Make up 1-2% of pancreatic tissue -Comprise ⍺, β and 𝛿 cells that secrete hormones

Question 455

What hormones do each cell in the islets of langerhans secrete?

Answer 455

⍺ = Glucagon β = Insulin 𝛿 = Somatostatin

Question 456

Name some regulators that increase insulin secretion?

Answer 456

-Glucose (MAJOR) -Amino acids (MINOR) -Neural cholinergic input (MINOR) -Gut hormones eg GLP1, GIP (MINOR)

Question 457

Name some regulators that decrease insulin secretion?

Answer 457

-Adrenaline -Somatostatin

Question 458

Name some regulators that increase glucagon secretion?

Answer 458

-Amino acids (MAJOR) -Stress neural input (MINOR) -Adrenaline (MINOR) -Cortisol (MINOR)

Question 459

Name some regulators that decrease glucagon secretion?

Answer 459

-Glucose (MAJOR) -Insulin (MAJOR) -Gut hormones eg GLP1/GIP (MINOR)

Question 460

Give the sequence of events in insulin production

Answer 460

-Preproinsulin is produced, a linear polypeptide consisting of a signal peptide, B-chain, C-peptide and A-chain -Proinsulin is produced by cleaving signal peptide, consisting of the B-chain, C-peptide, and A-chain. The A and B chain are linked by disulphide bonds -Mature insulin is produced, consisting of the A and B chains linked by two disulphide bonds

Question 461

Describe the insulin molecule and its storage

Answer 461

-Consisting of the A and B chains linked by two disulphide bonds, excluding an intra-chain disulphide bond in the A chain -In storage granules, insulin forms hexamers stabilised by zinc ions (this is biologically inactive) -Active in monomer form in bloodstream

Question 462

What is the use of C-peptide from insulin?

Answer 462

-Byproduct of insulin synthesis -Not degraded by the liver -Means it is valuable as a marker for assessing pancreatic beta cell function and insulin secretion

Question 463

Describe β cellular configuration in islets during a basal blood glucose concentration

Answer 463

-ATP K+ channel open, with K+ flux -Membrane hyperpolarised to -60mV -Ca2+ channel closed -Low insulin secretion

Question 464

What acts as the "glucose sensor" in β islet cells?

Answer 464

-Glucokinase activity -ie the amount of ATP in cell

Question 465

Describe β cellular configuration in islets during an increased blood glucose concentration

Answer 465

-Raised ATP -ATP K+ channel closed -Membrane depolarised to -40mV -Ca2+ channel open, with high Ca2+ -High insulin secretion

Question 466

What is MODY?

Answer 466

Maturity onset diabetes of the young

Question 467

What is PNDM?

Answer 467

Permanent Neonatal Diabetes Mellitus

Question 468

What is PHHI?

Answer 468

Persistant Hyperinsulinaemic Hypoglycaemia of infancy

Question 469

What mutations cause PNDM?

Answer 469

-KCNJ11 -ABCC8 -INS

Question 470

Name some insulin sensitive tissues

Answer 470

-Liver -Muscle -Adipose

Question 471

Describe what happens in each insulin-sensitive tissue postprandially

Answer 471

Liver - Glucose converted to glycogen and triglyceride Muscle - Glucose to glycogen or metabolised by glycolysis Adipose - Glucose converted to triglyceride

Question 472

What happens to dietary glucose when the liver glycogen stores are full?

Answer 472

-Converted to fat (de novo lipogenesis) -Increased lipogenesis in adipose tissue -Utilised for immediate energy needs -Very low density lipoprotein is secreted (VDLD)

Question 473

Describe what happens in each insulin-sensitive tissue post absorptive (fasted)

Answer 473

Liver - Produce glucose (from glycogen) and ketone bodies (from fatty acids) Muscle - Protein to amino acids (for gluconeogenesis) and metabolise fatty acids for fuel Adipose - Triglyceride converted to fatty acids and glycerol

Question 474

Describe the action of insulin on metabolic pathways in the liver

Answer 474

+Increase glycogen synthesis +Fatty acid/Triglyceride synthesis +Protein synthesis -Decreased glycogen degradation -Gluconeogenesis

Question 475

Describe the action of insulin on metabolic pathways in muscle tissue

Answer 475

+Increased glucose transport +Increased glucose oxidation +Increased glucose synthesis +Increased protein synthesis

Question 476

Describe the action of insulin on metabolic pathways in adipose tissue

Answer 476

+Increased glucose transport +Increased Triacylglycerol synthesis -Decreased Triacylglycerol breakdown release of fatty acids

Question 477

Describe the cellular action of insulin in muscle tissue

Answer 477

-Translocation of GLUT4 from vesicles to PM -Activation of glycogen synthase -Activation of pyruvate dehydrogenase

Question 478

Describe the cellular action of insulin in adipose tissue

Answer 478

-Increased translocation of GLUT4 from vesicles to PM -Increased fatty acid synthesis -Increased A-CoA carboxylase and fatty acid synthase -Decreased hormone sensitive lipase (HSL) -Increased TAG synthesis

Question 479

Describe the cellular action of insulin in the liver

Answer 479

-Activation of glycogen synthase and A-CoA carboxylase (covalent modification) -Inactivation of Phosphorylase (covalent modification) -Induced transcription of Glucokinase, A-CoA carboxylase, and fatty acid synthase -Repressed transcription of G6Pase, and PEPCK

Question 480

Describe PH protein domains

Answer 480

-Pleckstrin Homology domain -Binds to phosphorylated inositol phospholipid in plasma membrane -Involved in Insulin signalling pathway

Question 481

Describe PTB protein domains

Answer 481

-Binds phosphotyrosine (P-Y) residues -eg in Insulin receptor

Question 482

Describe SH2 protein domains

Answer 482

-Src Homology 2 domain -Binds phosphotyrosine residues surrounded by unique protein sequences -Involved in insulin signalling pathway as its recognises phosphorylated tyrosine residues (from the insulin receptor's intrinsic tyrosine kinase)

Question 483

Describe SH3 protein domains

Answer 483

-Src homology 3 domain -Binds specifically to proline-rich regions (eg SOS) -Links insulin receptor signalling to the activation of Ras/MAPK pathway

Question 484

Describe the insulin receptor

Answer 484

-Receptor Tyrosine Kinase (RTK) -Heterotetramer, made of ⍺⍺ββ subunits -Consists of Kinase activation loops, juxtamembrane domains, C terminal domain, Ser/Thr domains

Question 485

What is the function of each insulin receptor subunit

Answer 485

⍺ - Insulin binding domain β - Transmembrane and tyrosine kinase

Question 486

Give the functions of each Insulin receptor domain

Answer 486

Kinase activation loop - Activation of tyrosine kinase, phosphorylation creates binding site for SH2 Juxtamembrane domain - Phosphorylation creates binding site for PTB C terminal domain - Regulates IR kinase activity and kinase-adapter protein interactions Ser/Thr - Inhibits receptor kinase activty

Question 487

What follows insulin binding the the ⍺ subunit of Insulin receptor?

Answer 487

1 - Transphosphorylation of β units 2 - Activation of tyrosine kinase activity 3 - Phosphorylation of IR generates binding sites for proteins with PTB and SH2 domains

Question 488

What is SHC in insulin signalling?

Answer 488

-SH2 containing adapter protein -Links activated insulin receptor to Ras/MAPK pathway

Question 489

Describe the structure of Shc (in insulin signalling)

Answer 489

Consists of -PTB domain -CH1 domain -SH2 domain

Question 490

Describe Insulin receptor substrate

Answer 490

-Adapter proteins that serve as key adaptor molecules in insulin signalling pathway -Docking platforms propagating signals from activated insulin receptor -Functions include metabolic regulation, growth and proliferation, tissue specific roles

Question 491

Name the genes that encode insulin receptor substrate, and key types of insulin receptor substrate

Answer 491

-IRS1, IRS2, IRS3 and IRS4 -IRS1 primarily involved in metabolic effects eg glucose uptake and glycogen synthesis -IRS2 primarily involved in metabolic and growth pathways and is critical for pancreatic β cell function

Question 492

What domains do insulin receptor substrates contain?

Answer 492

PH domain and a PTB domain, enabling binding to phosphorylated insulin receptor

Question 493

Describe signalling to the MAP-Kinase pathway via GRB2 in the insulin signalling pathway

Answer 493

-Grb2 binds to phosphorylated IRS or Shc through SH2 -This activates and uncovers SH3 domain -Which binds to SOS, which acts as a GDP/GTP exchange factor -Ras is activated when bound to GTP -This activates Raf, which through a cascade activates MAP-Kinase

Question 494

What is the function of MAP-Kinase/ERK in the insulin signalling pathway?

Answer 494

Drives growth, differentiation, and proliferation

Question 495

Describe signalling to the PI-3-Kinase pathway in the insulin signalling pathway

Answer 495

-Insulin binds to IR⍺ subunit, leading to phosphorylation of IRS on tyrosine -Leads to recruitment of proteins (eg PI3K) to IRS via SH2 domains causing formation of PIP3 -PIP3 recruits AKT/PKB and PDK1 to plasma membrane -Dual phosphorylation and activation of AKT/PKB -Leading to phosphorylation of Protein kinase B/AKT substrates

Question 496

Describe how insulin binding to Insulin receptor ⍺ subunit leads to phosphorylation of Insulin receptor substrate on tyrosines

Answer 496

-Insulin binds to subunit -Activating IR tyrosine kinase -Autophosphorylation of tyrosines -Recruitments of IRS to IR -Phosphorlation of IRS on tyrosines

Question 497

Describe phosphatidylinositol 3-kinase (PI3K)

Answer 497

-Adds a phosphate group to the 3 position of the inositol ring in phosphatidylinositol, creating PIP3 -Consists of two subunits, being p110 (catalytic) and p85 (regulatory)

Question 498

What is the functions of PIP3?

Answer 498

-Insulin signalling -Cell proliferation -Apoptosis -Cell motility -Immune activation

Question 499

What mediates the degradation of PIP3?

Answer 499

PTEN (phosphatase and tensin homologue)

Question 500

What are mutations in PTEN associated with?

Answer 500

Cancer and other diseases as it is a tumour suppressor gene (as well as being involved in insulin signalling)

Question 501

What domains make up AKT/protein kinase B?

Answer 501

-N terminal Pleckstrin homology domain (PH) -Central kinase domain -C terminal Hydrophobic regulatory domain

Question 502

What activates AKT/Protein kinase B?

Answer 502

Dual Phosphorylation (by PIP3) on Thr308 (PKD1) and Ser473 (mTORC2)

Question 503

Give some examples of what AKT/Protein kinase B phosphorylates?

Answer 503

-Glycogen synthase kinase 3 (inactivating) -Preventing inhibition of glycogen storage, lipid synthesis and protein synthesis -AS160 (involved in GLUT4 translocation) -FOXO1 -BAD

Question 504

Describe the action of AKT/Protein Kinase B phosphorylation AS160

Answer 504

-Inactivates it -Causing Glut4 translocation to plasma membrane -Glut4 is the main glucose transporter in muscle and adipose tissue

Question 505

Describe the action of AKT/Protein Kinase B in protein synthesis

Answer 505

-mTORC1 is major regulator of protein synthesis, and is regulated by TSC1:TSC2 complex (inhibits Rheb by converting GTP to GDP) -AKT phosphorylates TSC2, inhibiting it -Rheb-GTP activates mTORC1 -mTORC1 activates elF4E-BP1 and S6K1 leading to translation initiation and ribosome biogenesis

Question 506

Describe the action of AKT/Protein Kinase B on FOXO1

Answer 506

-FOXO1 acts as a transcription factor inducing G6PC and PEPCK (gluconeogenesis enzymes) and represses Glucokinase -AKT increases degradation of FOXO1

Question 507

Describe what causes type 1 diabetes

Answer 507

-Polygenic disorder -Leading to autoimmune detruction of the insulin producing cells

Question 508

Describe what causes type 2 diabetes

Answer 508

-Polygenic disorder -Defects in insulin action (obesity) -Defects in glucose induced insulin secretion

Question 509

Give some complications found in diabetes patients

Answer 509

-Retinopathy (loss of sight) -Nephropathy (kidney function) -Peripheral neuropathy (loss of feeling in feet) -Autonomic neuropathy (affecting cardiovascular, gut and urinary functions) -Macrovascular complications ALL CAUSED BY CHRONIC HYPERGLYCAEMIA

Question 510

Why is type 2 diabetes associated with obesity?

Answer 510

-Obesity is associated with insulin resistance and compensatory enlargement of islets -Genetic background determines the extent by which β cels can compensate -Type 2 diabetes develops once β cell capacity can no longer compensate for insulin resistance

Question 511

What does increased insulin resistance lead to in those with robust β cells?

Answer 511

-Increased insulin secretion (compensation) -Leading to -Hyperinsulinaemia -Euglycaemia (GOOD)

Question 512

What are the two types of therapy for type 2 diabetes?

Answer 512

-Lifestyle changes (decreasing calorie intake and increasing exercise) -Drugs (either monotherapy or combination therapy)

Question 513

What are the main targets of drugs for type 2 diabetes

Answer 513

-Carbohydrate digestion -Renal glucose excretion -Insulin secretion -Adipose tissues -The liver

Question 514

Describe how ⍺-glucosidase inhibitors are used to treat type 2 diabetes, and give an example

Answer 514

-Slow down digestion and absorption of carbohydrates -Reduce rate at which complex carbohydrates are broken down into glucose by glucosidases -Slow the rise in postprandial blood glucose EXAMPLE: Miglitol

Question 514

Give some benefits and drawbacks of ⍺-glucosidase inhibitors as treatments for Type 2 diabetes

Answer 514

+ Decrease intestinal glucose absorption + Decrease glycemic index of food + Decrease post prandial blood glucose conc + Decrease post prandial triacylglycerides + No risk of hypoglycaemia - Causes abdominal discomfort - Fermentation of undigested carbohydrate in the colon

Question 515

Describe how SGLT inhibitors are used to treat type 2 diabetes, and give an example

Answer 515

-Target SGLTs in the kidneys -Prevent reabsorption of glucose, causing excretion in urine EXAMPLE: Phlorizin

Question 516

Give some benefits and drawbacks of SGLT inhibitors as treatments for Type 2 diabetes

Answer 516

+ Can cause weight loss due to loss of calories in urine + Slow the progression of chronic kidney disease by reducing kidney workload - Increased urinary volume - Risk of urinary tract infections (due to more sugary environment) - Risk of genital fungal infections (due to more sugary environment)

Question 517

Describe how Sulphonylureas are used to treat type 2 diabetes

Answer 517

-Bind to SUR1 in K+/ATPase channel -Acting as an antagonist, closing the channel, depolarising the membrane in islet cells, leading to insulin secretion

Question 518

Give some benefits and drawbacks of sulphonylureas as treatments for type 2 diabetes

Answer 518

+ Decrease blood glucose + Increase insulin secretion independently of blood glucose - Increased risk of hypoglycaemia - Increased weight gain - Increased risk of cardiovascular events

Question 519

How are GLP1R agonists and dipeptidyl peptidase-4 inhibitors (as treatments for type 2 diabetes) similar?

Answer 519

Target incretins

Question 520

What are incretins, and give some examples

Answer 520

-Intestinal peptides produced in response to food that stimulate insulin secretion -GIP (glucose dependent insulinotropic peptide) -GLP1 (glucagon like peptide 1)

Question 521

Where are GLP1/GIP receptors expressed, and what is their action?

Answer 521

-Pancreas (Increase insulin secretion, either decrease or increase glucagon) -Gut (GLP1 can lead to decreased gastric emptying) -Kidney (GLP1 can decrease Na excretion) -Brain (Decrease caloric intake)

Question 521

Give the mechanism of action of GLP1 agonists on the islet β cells

Answer 521

-Increase cAMP, leading to increased activity of protein kinase A, increased insulin secretion as K+/ATPase channel is shut, forcing depolarisation -Increased PI3K, leading to activation of AKT/PKB, increasing gene transcription, decreasing apoptosis, and stimulating cell growth

Question 522

What degrades incretins (and describe them)?

Answer 522

-DPP4 (dipeptidyl peptidase 4) -Exist either as membrane-anchored extracellular enzyme and in soluble form

Question 523

Describe how DPP4 inhibitors are used to treat type 2 diabetes, and give an example

Answer 523

-Prevent breakdown of incretins -Allowing them to inhibit glucagon secretion and increase insulin secretion, lowering blood glucose EXAMPLE: Sitagliptin

Question 524

Give some benefits and drawbacks of GLP1R agonists and DPP4 inhibitors as treatments for type 2 diabetes

Answer 524

+ Moderately decrease blood glucose + Increase insulin secretion dependently of blood glucose + Decrease glucagon secretion + No risk of hypoglycaemia + Decrease food intake and body weight - Potential risks for pancreatitis or pancreatic cancer - Cause gastric discomfort

Question 525

Describe what occurs to adipose tissue in obesity

Answer 525

-Fat cell hypertrophy (increase in cell volume) -Fat cell hyperplasia (increase in cell number)

Question 526

What occurs when adipocytes attain their maximum capacity of lipid storage?

Answer 526

Lipid levels are raised in blood, liver, muscle and islet β cells

Question 527

Describe how PPARγ drugs are used to treat type 2 diabetes, and give an example

Answer 527

-Favour adipocyte proliferation and further lipid storage (rather than raising lipid levels in blood and other tissues), as well as increasing lipogenesis and sensitivity to insulin -Preventing damage by lipids in other organs EXAMPLE: Thiazolinediones

Question 528

Give some benefits and drawbacks of PPARγ drugs as treatments for type 2 diabetes

Answer 528

+Chronically lower blood glucose and insulin +Increases insulin sensitivity in obesity ~Increases bodyweight and adiposity -Only pioglitazone still in use as others increased liver damage or chance of heart attacks

Question 529

How does metformin work as a treatment for type 2 diabetes

Answer 529

-Inhibition of hepatic gluconeogenesis -Increased insulin sensitivity in liver and periphery -Increases peripheral glucose uptake and utilisation -Increases fatty acid oxidation

Question 530

Describe the cellular mechanisms by which metformin decreases gluconeogenesis and increases fatty acid oxidation

Answer 530

-Metformin enters cells via OCT1, and accumulates in energised mitochondria -This inhibits complex I, decreasing ATP and ADP, increasing AMP, leading to AMPK activation -AMP kinase inhibits PEPCK and G6Pase, inhibiting gluconeogenesis -AMP kinase also inhibits A-CoA carboxylase, increasing fatty acid oxidation and decreasing fatty acid synthesis