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Year 4 > M&C Pharmacology > Flashcards

M&C Pharmacology Flashcards

1
Q

Example ionotropic receptor
Example kinase linked receptor
Example GPCR (metabotropic)
Example nuclear receptor

A

Nicotinic acetylcholine receptor
PDGF receptor
Muscarinic acetylcholine receptor
Steroid hormone receptors

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2
Q

What are the 3 families of GPCRs and examples of specific GPCRs in each family?

A

Family A: rhodopsin-like - e.g. retinal
Family B: glucagon-like - e.g. cacitonin
Family C: metabotropic glutamate-like - e.g. glutamate

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3
Q

What is the GRAFS system and what does it stand for?

A

Another grouping system for GPCRs, stands for: glutamate, rhodopsin, adhesion, frizzled, secretin

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4
Q

What are RAMPs and what do they do?

A

Receptor activity modifying proteins (RAMPs) selectively interact with some family B GPCRs modifying their pharmacological properties (e.g. CRLR) - which RAMP expressed determines which ligand binds

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5
Q

Give example ionotropic receptors, some that are trimers, tetramers, pentamers

A

Trimer - P2X receptor
Tetramer - NMDA receptor, AMPA receptor, Kainate receptos
Pentamer - Nicotinic acetylcholine receptor, 5HT3 receptor, GABAa receptor, glycine receptor

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6
Q

What are the two chemically distinct classes of calcium channel blocker that have preferential effects on heart vs vasular smooth muscle?

A

Phenylalkylamines are charged - effective in the heart (cardiac dysrhythmias) e.g. verapamil
Dihydropyridines are neutral - effective in vascular smooth muscle (hypertension - vasodilation) e.g. nifedipine

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7
Q

What is the Gby subunit important for?

A

Receptor recognition (dissociation inhibitor), GDP-bound Ga-subunit recognition, effector recognition

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8
Q

What are the different Ga subunit subfamilies and what do they do?

A

Gas - adenylyl cyclase activation
Gai/o - adenylyl cyclase inhibition, ion channel modulation
Gaq/11 - PLC activation
Ga12/13 - scaffolds for regulators of monomeric GTPases

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9
Q

What are the GPCR second messengers and what pathway do they catalyse?

A

Adenylyl cyclase - ATP -> cAMP + PPi
PLC - PIP2 -> IP3 + DAG
PI3K - PIP2 -> PIP3

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10
Q

What are RGS proteins and what do they do?

A

Regulator of G-protein signalling (RGS) - work primarily by accelerating the intrinsic GTPase activity of Ga subunits causing more rapid switching off of G-protein signalling

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11
Q

What are GIRKs and what do they do?

A

G-protein regulated inwardly rectifying K channels (GIRKs) - Gβγ dimeric protein interacts with GIRK channels to open them so that they become permeable to potassium ions, resulting in hyperpolarization of the cell membrane

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12
Q

What are the roles of proteins possessing RH domains?

A

p115rho/gef allows specific GPCRs to activate the monomeric GTPase Rho.

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13
Q

What are the key regions of GPCRs which give rise to CAMs?

A

TMe/i2 interface
Membrane proximal regions of the i3 loop
TM6/e4 interface

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14
Q

How can you study GPCR conformational changes?

A

FRET

Crystallography overlays

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15
Q

What type of channel are GABAa receptors?

A

Ligand gates Cl- conducting ion channels (inhibitory transmission in the CNS)

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16
Q

How can we study the binding reaction of a ligand to a receptor?

A

Radioligand binding procedure:

1) Choose and make tissue/cell preparation containing receptor
2) Select suitable radiolabelled ligand
3) Incubate receptor preparation with appropriate concentrations of labelled ligand for a defined time and temperature - needs to reach equilibrium
4) Separate and count bound and free radioligand
5) Repeat steps 3 and 4 with addition of unlabelled ligand or modulatory agent
6) Analyse data to extract quantitative estimates of Bmax and Kd of labelled and unlabelled ligand

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17
Q

What are 3 effectors in cAMP mediated signalling?

A

PKA
Cyclic nucleotide gated channels
Cyclic nucleotide regulated GEFs

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18
Q

Example PKAII localisation

A

Attached to AKAPs localising it to SERCA and phospholamban - allows it to phosphorylate phospholamban which reduces inhibition on SERCA allowing increase calcium entry into SR in heart cells

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19
Q

How does coffee wake up your cells?

A

Caffeine blocks A2a receptor which reduces phosporylation of DARPP-32 at T34 allowing phosphatase I to dephosphorylate phosphorylated targets. It also prevents DARPP-32 dephosphorylation at T75 which means DARPP-32-T34P inhibits PKA

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20
Q

What is Epac and what does it do?

A

Exchange protein directly activated by cAMP - functions as GEFs for Ras-like small GTPases Rap1 and Rap2 independently of PKA (e.g. Epac is activated by cAMP from glucagon-like peptide I binding to GLP-IR and this causes the pancreas to secrete insulin in from pancreatic B-cells)

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21
Q

What are 3 effectors in cGMP signalling?

A

Cyclic nucelotide gated channels
cGMP dependent PKG
Modulation of PDE activity

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22
Q

What are the 3 groups of particulate guanylyl cyclases based on ligand specificity?

A

Netriuretic peptide receptors
Intestinal peptide-binding receptors
Orphan receptors

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23
Q

What are soluble guanylyl cyclases activated by?

A

NO

Carbon monoxide

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24
Q

What are the 3 types of NOS and their functions?

A

iNOS - inducible, produces high NO concentration that can exhibit direct toxic effects. Immune and cardiovascular systems - immune defence
eNOS - endothelial - vasodilation
nNOS - neuronal (+skeletal muscle) - communication

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25
Q

Biosynthesis pathway of NO?

A

L-arginine -> citrulline + NO

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26
Q

How does smooth muscle contration and relaxation come about?

A

Calcium enters the cell and forms complexes with calmodulin. Calcium-calmodulin complex activates MLCK which phosphorylates MLC which increases ATPase activity causing contraction. Relaxation: MLCP dephosphorylates MLC causing ATPase actvity to be reduced.

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27
Q

How does thromboxane A2 work?

A

Acts through GPCR Ga12/13 to increase RhoA which increases ROCK which inhibits MLCP and so prevents relaxation (vasoconstrictor)

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28
Q

Vasodilation pathway through eNOS

A

Shearing forces and muscarinic receptors trigger intracellular calcium increase which triggers eNOS to make NO which diffuses into smooth muscle cell immediately adjacent to endothelium and activates guanylyl cyclase to generate cGMP. cGMP interacts with PKG which opens K channels causing K to leave to cell causing hyperphosphorylation which reduces calcium channel open probability so less calcium enters the cell so less contraction takes place
PKG also phosphorylates RGS2 which accelerates hydrolysis of Gq which turns down signalling by Gq coupled receptors therefore lowering calcium entry and so intracellular calcium decreases
Fall in calcium inactivates MLCK
PKG can also phosphorylate IRAG which is IP3 receptor associated protein causing dissociation of IRAG reducing activity of IP3 resulting in less calcium release
PKG also phosphorylates MLCP which allows the cell to relax

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29
Q

What do PDEs do?

A

Hydrolyse cGMP and/or cAMP

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30
Q

What is the erection pathway?

A

Psychogenic/reflexogenic stimuli cause neural depolarisation which causes an increase in intracellular calcium which binds to calmodulin. Calcium-calmodulin complex triggers nNOS which produces NO. NO diffuses into cells and activates soluble gunaylyl cyclase which catalyses GTP to cGMP. cGMP can now undergo 2 pathways: it can activate PKG which decreases intracellular calcium concentration which causes corpus cavernosum smooth muscle relaxation which leads to inflow of blood and therefore erection OR: PDE5 hydrolyses cGMP to 5’GMP (degrading it)

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31
Q

How does viagra work?

A

Binds to catalytic sites on PDE5 competing with cGMP which maintains PKG levels

32
Q

Other than for erectile dysfunction, what clinical use does PDE5 inhibitors have?

A

Preferentially dilate vessels in well oxygenated areas of the lung (i.e. rematch conditions of vasodilation)

33
Q

What are some functions of Akt?

A

Promotes cell survival by inhibitinf apoptosis
Turns off catalytic activity of GSK3 resulting in activation of pathways normally repressed by this enzyme (promotes proliferation, migration etc)
Blocks transcription of genes normally stimulated by forkhead-related TF1

34
Q

How is PI3Ksignalling switched off?

A

Degradation of PIP3 by at least 2 different phosphatases
SHIP1 (restricted to meatopoietic cells)
SHIP2 (ubiquitous expression)
- SHIP activity removes binding sites for proteins with PIP3 selective PH domain but generates PIP2 that PKB/Akt can bind to
PTEN (phosphatase)
- removes both PIP3 and PIP2 which inhibits PKB/Akt

35
Q

What is the core structure of all PLCs?

A

PH (binds PIs), EF (function unclear), X + Y (catalytic domains), C2 (calcium binding)

36
Q

How can PLC-y be activated?

A

Activated RTKs bind PLC-y
Non-RTKs are recruited by e.g. cytokine receptors and recruit effectors with SH2 domains e.g. PLC-y
GPCRs can transactivate RTKs by e.g. Src related non-RTKs and activate PLC-y

37
Q

How can you monitor the activity of PLC?

A
  1. Changes in [calcium]i (single cell populations) or functionl consequence of change
  2. Accumulation of [3H]-labelled inositol phosphates against lithium block of inositol monophosphate activity - total PLC activity independent of IP3 metabolism
  3. Mass of IP3 - using radioreceptor assay
  4. Single cell imaging of PLC activity - biosensor for IP3/PIP2 uses a PH domain tagged with a molecule of enhanced GFP
38
Q

What is the intra and extracellular calcium concentrations at rest?

A 
EC = 1-2mM
IC = 100nM
39
Q

How do cells regulate their [calcium]i?

A
  • Relative impermeability of the plasma membrane
  • Expulsion of calcium across the plasma membrane via: calcium ATPase ([Ca]i increases, Ca binds to calmodulin, Ca-calmodulin binds Ca-ATPase, Can-ATPase removes Ca) AND: NCX (Na gradient used as driving force)
  • Calcium buffers
40
Q

Describe ER store refilling via STIM1

A

STIM1 sits on ER membrane and has a calcium sensor (binds calcium and changes conformation based on how much is bound). When store is empty, calcium no longer binds to STIM1 allowing it to coalesce and interact with CRAC channels in the plasma membrane. This opens CRAC channels and allows calcium in right next to the ER. SERCA pumps can then pump calcium into the ER.

41
Q

How was G-protein independent signalling first demonstrated?

A

Dictylostelium discoideum - exists mainly as single cell entity moving around consuming bacteria. When it runs out of bacteria it goes into starvation mode where multiple entities congregate together to form fruiting bodies. These give off spores which land somewhere with more bacteria. Relies to some extent on GPCR signalling but has depleted complement of G protein subunits, only 1 Beta subunit - look at what happens when you remove this - still signals therefore the GPCR must signal independently of G proteins

42
Q

How was it found that desensitisation occurs at the level of the receptor?

A

cyc- lymphoma cells lack Gs protein. Desensitise protocol then reconstitute with WT lysate containg Gs proteins - still see desensitisation even without signlling pathway

43
Q

How do GPCRs desensitise?

A

Desensitisation correlates with phosphorylation. (e.g. by PKA or GRKs). Arrestin binds to phosphorylated receptor and causes clathrin mediated internalisation which moves receptor-arrestin complex into endosome where its fate is decided. pH in endosome more acidic than the slightly alkaline cytoplasm. The acidic environment facilitates dissociation of ligand from receptor. Also changes conformation of receptor favouring arrestin dissociation and dephosphorylation of receptor. The receptor can either be recycled to membrane or degraded - method of adjusting number of receptors available to signal

44
Q

What might determine cell specific receptor-arrestin signalling?

A

Conformation of arrestin (active conformation of arrestin cn determine which signalling pathway the protein scaffolds with)

45
Q

Describe the phototransduction pathway

A

Rhodopsin (GPCR opsin and covalently bound prosthetic group 11-cis-retinal) is present in high concentrations in the retinal rod outer segment discs. A photon of light causes isomerisation of 11-cis-retinal to all-trans-retinal, conformational change in rhodopsin and increased its GEF activity towards G protein transducin. Activated transducin interacts with PDE6 sequestering inhibitory PDEy subunits and unmasking the full catalytic activity of PDEaB. PDEaB hydrolyses cGMP leading to closure of cGMP regulated cation channels which causes decreased sodium influx - hyperpolarisation and decreased IC [calcium]

46
Q

What is needed for recovery following activation of the phototransduction cascade?

A
  1. Inactivation of rhodopsin: activated rhodopsin is a substrate for rhodopsin kinase, multiple phosphorylations of the C terminal serine residues of rhodopsin occur creating high affinity binding site for rod arrestin.
  2. Inactivation of transducin-GTP: RGS9-1 (GAP)
  3. Re-synthesis of cGMP: guanylyl cyclase is not directly calcium sensitive but is regulated by the calcium binding protein GCAP. On illumination [calcium]i decreases, calcium dissociates from GCAP releasing it to exert its GC activating activity and increasing the rate of cGMP synthesis
47
Q

What are the regulatory mechanisms for adaptation of rod cell phototransduction?

A

Changes in the basal [calcium]i in the rod outer segment affects the activities GC, rhodopsin kinase and cGMP regulated cation channels through calcium sensitive regulatory proteins
Changes in the basal phosphorylation of rhodopsin
Translocation of key phototransduction components between ros outer and inner segments (e.g. as ambient light levels change the subcellular distribution of transducin and arrestin change rapidly and reciprocally)

48
Q

What calcium sensitive regulatory proteins are involved in photo-adaptation?

A

Recoverin, GCAP and calmodulin: In its calcium bound form, recoverin binds tightly to rhodopsin kinase (GRK1) preventing it from phosphorylating rhodopsin. In its calcium bound form, GCAP is inactive. In its calcium bound form, calmodulin binds and inhibits the cGMP regulated cation channel.

49
Q

What is phosphate counting in rhodopsin phosphorylation?

A

Evidence suggests more than one C terminal site on rhodopsin must be phosphorylated by GRK1 before arrestin binds. In dark adapted rod cells, most rhodopsin is unphosphorylated, whereas with increasing levels of background illumination, rhodopsin may be in its mono or diphosphorylated form.

50
Q

How does adjusting the levels of key signalling components within the outer segment facilitate adaptation in phototransduction?

A

On prolonged light exposure, arrestin and RGS9-1 translocate into the outer segment while transducin and recoverin translocate out of the OS. This permits change in the ability of the phototransduction cascade to amplify and efficiently shut off signalling.

51
Q

What is the purpose of phototransduction adaptation?

A

Allows ability to detect and report visual stimuli over a wide range of levels of background illumination

52
Q

Give some evidence for the caveolae/raft signalling hypothesis

A

In cardiac myocytes, B-adrenergic receptors located in caveolae efficiently activate adenylyl cyclase 6 while prostaglandin E2 receptors (not in caveolae) do not (AC6 is solely localised in caveolae)

53
Q

Give an example of a complex recruited to activated receptor an a stable complex

A

Recruited: PDGFR
Stable: NMDA receptor-yotiao complex

54
Q

What do the following protein interaction domains bind to?
SH2
SH3
PH
PTB
PDZ
Why is it useful to know a protein’s structure?

A

SH2: binds to short peptide motifs containing pY followed by 3-6 amino acids C terminal to pY
SH3: binds to proline rich peptide sequences
PH: binds to PIs such as PIP2 and PIP3
PTB: binds to motifs containing pY and 3-8 amino acids N-terminal to pY
PDZ: binds to last 4-5 C terminal residues in target proteins, typically ion channels and receptors
You can predict protein-protein interactions

55
Q

Give 2 drugs that modify protein-protein interactions

A
  1. Immunosuppressive drug FK506 inactivates calcineurin by creating an unphysiological complex between calcineurin and FKBP12. Inactivation of calcineurin interrupts signals from the immune systems T cell receptor.
  2. Rapamycin brings together 2 proteins that normally ignore each other (FKBP12 and FRAP). FRAP normally turns on a ribosomal kinase responsible for protein synthesis, but fails to do so when linked to rapamysin and FKBP12. This inhibits proliferation of the immune system’s T lymphocytes
56
Q

What does ERK phosphorylate in the cytoplasm?

A

PLC A2
p90 ribosomal S6 kinase
A-type K channels

57
Q

How do GPCRs couple to MAPK signalling?

A
  • Ga or Gby dependent and involve conventional signalling intermediates
  • Some GPCRs can transactivate RTKs to act as scaffolds for Grb2/mSos recruitment and ras activation
  • Some nRTK such as src family kinase pyk2 and FAK are intermediates in MAPK activation by GPCRs in some cells
  • GPCR activation can result in proteolytic cleavage of cell surface tethered growth factor often via MMPs. The released growth factor can then act as an autocrine/paracrine activator of RTKs
58
Q

What are the 3 MAPK pathways?

A

Growth factors -> receptor -> Raf -> MKK -> ERK -> growth/differentiation
Cellular stress/cytokines -> receptor -> MEKK, MLKs, ASK -> MKK -> JNK
OR
-> MEKK, MLK, ASK -> MKK -> p38 -> apoptosis, inflammation, differentiation

59
Q

Give 3 examples of latent cytoplasmic transcription factors

A

Wnt/B-catenin (wnt binds to LRP and frizzled causing recruitment and phosphorylation (activation) of dishevelled which disrupts the Axin/APC/CKIa/GSK-3 complex leading to GSK-3 inhibtion which prevents B-catenin from being degraded so it accumulates in the cytoplasm and translocates to the nucleus where it displaces co-repressor groucho on transcription factor TCF and recruits co-activator to cause cell proliferation)
SMAD (TGFbeta binding causes TGFB receptor complex formation and activation of its type II kinase domain. Activated TGFBR type II kinase activates type I kinase domain which activates the SMAD2/3 protein which is in the cytoplasm near the membrane. SMAD4 forms a complex with 2xSMAD2/3 to form functional transcription factor. Translocates to nucleus and with the help of co-factors leads to gene transcription - causes cell growth, differentiation etc (SMAD7 blocks the formation of SMAD4-SMAD2/3 complex))
PI dependent Tubby (agonist binds to GPCR, Gq activation, activation of PLC-B, hydrolyses PIP2 -> DAG + IP3, tubby protein dislodged from membrane, nuclear translocation -> activates gene transcription)

60
Q

What are the morphological criteria of apoptosis?

A 
Rounding of cell
Retraction of pseudopodes
Reduction of cellular and nuclear volume
Nuclear fragmentation
Minor modification or cytoplasmic organelles
Plasma membrane blebbing
Engulfment by resident phagocytes
61
Q

What are the morphological criteria of necrosis?

A

Cytoplasmic swelling
Rupture of plasma membrane
Swelling of organelles
Moderate chromatin condensation

62
Q

What are the morphological criteria of cornification?

A 
(specific to skin - cells are gradually eliminated forming a dead lprotective barrier on skin surface)
Elimination of cytosolic organelles
Modifications to plasma membrane
Accumulation of lipids in the EC space
Desequamation by protease activation
63
Q

What are the morphological criteria of autophagy?

A

Lack of chromatin condensation
Massive vacuolisation of the cytoplasm
Accumulation of double membraned autophagic vacuoles
Little or no uptake by phagocytic cells

64
Q

How are phagocytes drawn in to degrade apoptotic cells?

A

Phosphatidylserine translocates to the outside of the cell

65
Q

If Katp channels are not voltage sensitive and act as a potassium pore in the membrane, why is the I-V relationship not linear?

A

Inward rectifier block by polyamines

66
Q

Give the tissue specific Katp channels

A

Kir6.2/SUR1 - pancreatic beta cells
Kir6.2/SUR2A - cardiac myocytes
Kir6.2/SUR2B - smooth muscle
Kir6.1/SUR2B - vascular smooth muscle

67
Q

Stimulus-secretion coupling in pancreatic beta cells:

A

Katp channels are open when EC glucose is low. EC glucose increases the beta cell metabolism that increases the IC ATP levels and Katp channels close. This results in membrane depolarisation and opening of voltage gated calcium channels. Influx of calcium triggers insulin exocytosis

68
Q

How can Katp channels be utilised to treat type II and type I mild diabetes?

A

Katp channel inhibitors (e.g. glibencamide, tolbutamide) cause membrane depolarisation, calcium channel opening and insulin release

69
Q

Which Katp channnel inhibitor is the best choice to target diabetes type II and why?

A

Tolbutamide - most specific

70
Q

What mutations cause mild type I diabetes?

A

Mutations in the ATP binding site, Katp channel has reduced sensitivity to ATP so that the channel favours the open state - consequence is permanent neonatal diabetes mellitus

71
Q

How can mutations in SUR1 nucleotide binding domains in class II hyperinsulinemic hypoglycemia of infancy be treated?

A

Katp channel openers e.g. diazoxide

72
Q

Other than diabetes, what are Katp channel openers clinically used for?

A

Decrease hypertension by opening Katp channels in smooth muscle cells (Katp channels open causing hyperpolarisation, calcium channels close, less calcium inside the cell - relaxation) e.g. nicorandil
Alopecia - Kir6.2/SUR1 and Kir6.1/SUR2B are expressed in hair follicles, Katp channel opener minoxidil activates hair growth (molecular mechanism unknown)

73
Q

Define hirsuitism and hypertrichosis

A

Hirsuitism - inappropriate hair growth in the normal pattern of growth
Hypertrichosis - excessive hair growth outside the noraml pattern of hair growth

74
Q

What are 2 clinical uses for 5-HT3 receptor antagonists?

A

Emesis

GI function

75
Q

What are the 2 ways the vomiting centre can be activated?

A

Chemoreceptor trigger zone (CTZ) - not protected by blood-brain barrier allowing a chemosensory role. CTZ can be stimulated by toxins in the blood or triggered by neuronal input from the higher centres of the brain. CTZ releases 5-HT in the neuro-circuitry linking to vomiting centre to trigger the vomiting reflex
GI tract - 5-HT released from enterochromaffin cellsof the intestinal mucosa stimulated by luminal stimuli e.g. food poisoning. This results in the stimulation of peripheral 5HT3 receptors on vagal afferents. This vagal afferent then stimulates local releaseof 5HT in the neuro-circuitry of the brain stem in the vomiting centre which triggers the vomiting reflex

Year 4 (6 decks)

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