Pharmacology Flashcards
1
Q
State Signalling molecules
A
- Gases: nitric oxide and carbon monoxide.
- Amino acids: glutamate and glycine
- Small peptides: growth factors (enkephalins, NGF and BDNF).
- Proteins: insulin and glucagon, cytokines etc.
- Steroids: testosterone, cortisol
Neurotransmitters, Neuropeptides, Hormones, Inflammatory Mediators
2
Q
State Signalling types
A
- Passive diffusion from cell to cell.
- Direct cell-cell contact through gap junctions.
- Receptor mediated cell-cell signaling
How cells use these chemicals for communications
3
Q
How is Passive diffusion used for signalling?
A
• Small or hydrophobic molecules can cross plasma membranes.
• Nitric oxide is produced in endothelial cells which line the lumen of blood vessels.
• NO diffuses from endothelial cells to smooth muscle cells where it binds directly to the target enzyme which leads to relaxation of smooth muscle cells.
Nitric oxide, Viagra
4
Q
How are gap junctions used to signal?
A
- In heart muscle cells: synchronise muscle contraction.
- In the cochlea: mutations in connexins are one of the leading genetic causes of deafness.
They directly connect the cytoplasm of two cells, which allows various molecules, ions and electrical impulses to directly pass through a regulated gate between cells.
5
Q
How is Receptor mediated signalling used?
A
1) Synthesis
2) Secreted (exocytosis?) and transport (via blood, extracellular fluid)
3) Detection by a specific receptor on (or in) the target cell
4) Intracellular signalling molecules cascade the signal to target proteins
5) Signal(s) reach effector protein
6) Outcome
6
Q
Name two receptor types
A
Intracellular and Cell surface
7
Q
Give examples of cell surface receptors
A
- Contact-dependent receptors
- Ion channels
- G-protein coupled receptors
- Enzyme linked receptors
8
Q
Describe the Contact-dependent receptor-mediated signalling
A
- Ligand is not secreted
- Ligands and receptors are membrane proteins
- Communication with neighbouring cells and extracellular matrix.
- A membrane ligand (protein, oligosaccharide, lipid) and a membrane protein of two adjacent cells interact.
- A communicating junction links the intracellular compartments of two adjacent cells, allowing transit of relatively small molecules.
- An extracellular matrix glycoprotein and a membrane protein interact.
9
Q
Describe the Autocrine receptor-mediated signalling
A
- Very short distance: a cell signals to self or identical neighbouring cells.
- Signalling cell secretes a ligand which binds to own receptors.
- Reinforce a developmental “decision” by a cell.
- Cancer cells use autocrine signalling to stimulate their own survival and proliferation
- ATP is an autocrine signalling molecule/transmitter
10
Q
Describe the Paracrine receptor-mediated signalling
A
- Short distance: a cell signals to neighbouring cells.
- Signalling molecule does not diffuse far or is destroyed by enzymes in extracellular matrix.
Aspirin: co-ordinates activity (prostaglandins signal)
- It interferes with prostaglandins
- Can be of same or different types
Paracrine signaling is a form of cell-to-cell communication in which a cell produces a signal to induce changes in nearby cells, altering the behavior of those cells
11
Q
Describe the Endocrine receptor-mediated signalling
A
- Long distance: a cell signals to cells distributed widely in body.
- Signalling molecules are secreted into bloodstream e.g. hormones
12
Q
Describe the Synaptic signalling receptor-mediated signalling
A
- Used by neurons to communicate with neurons or other cell types.
- Ligand is called a “neurotransmitter”.
- Long or short distance, axons can be 1mm to 1m.
- Neurotransmitter moves a very short distance from target cell - the “synaptic cleft”.
Cell-cell communication
Always packaged into a vesicle
13
Q
Signalling Cascades- What determines the outcome of cell signalling?
A
- The complement of receptors the cell possesses.
- The complement of intracellular signalling molecules the cell possesses. This is known as the “intracellular signalling cascade”.
14
Q
What can Conformational change be detected by?
A
- Detected by intracellular signalling proteins (they make a link between receptor and effector)
15
Q
Name proteins used in intracellular signalling cascades
A
Relay proteins Adaptor Proteins Bifurcation proteins Transducer proteins Integrator proteins Latent gene regulatory proteins
16
Q
Define Relay proteins
A
- pass the message
- transducer receptor signal to effector
17
Q
Define Adaptor proteins:
A
- Adaptor proteins are essential molecules that influence signal transduction pathways, governing cross-talk and specificity.
- Adaptor proteins contain protein-binding motifs, which facilitate interactions between protein-binding partners and the generation of bigger signalling complexes.
- Don’t have signalling functions
- Creates a bridge
- Allows receptors expand repertoire of substrates
18
Q
Define Bifurcation proteins:
A
take the message to another signalling pathway.
19
Q
Define Transducer proteins:
A
converts the signal to another form.
20
Q
Define Integrator proteins:
A
- integrates signals from different signalling pathways before relaying a signal onward.
- Regulate by multiple signals
21
Q
Define Latent gene regulatory proteins:
A
- migrate to nucleus when activated.
- Switch on or off particular genes
- Protein-protein interactions rapidly switched on/off
22
Q
Why is one-one signalling limited?
A
Not very amplified
When exposed to a low concentration
- Few receptors activated
- Few interactions
23
Q
What are the advantages to amplification?
A
Signal strength can be amplified
When amplification is built in, a single receptor can lead to amplification
Very significant
Response ends up being very large from only a few
Cost effective
24
Q
How do Intracellular signalling cascades integrate signalling pathways?
A
There is cross-talk between receptors
One receptor switching off or sum up to produce a stronger signal
25
What are the effects of Intracellular signalling cascades?
- can have a positive feedback loop to amplify the signal
- can have a negative feedback loop to terminate the signal
- integrate signalling pathways.
26
Name three drugs that target ion channels
Lignocaine- a local anaesthetic
Apamin- in a bee’s sting
Dihydropyridines- Eg. nifedipine
27
Name three drugs that target enzymes
► Acetylsalicylic acid
► Pargyline and clorgyline
► Captopril
28
Name two drugs that target transporters
- Fluoxetine- Prozac (anti-depressant)
| - Digoxin
29
What else could be used as a drug target?
Receptors involved in cell-cell communication- their effects on cell signalling will depend on whether they are agonists or antagonists
30
How can receptors be classified?
by their structure
31
State the four classifications of receptors?
Type 1 ligand-gated ion channels - ionotropic
Type 2 G-protein coupled receptors - metabotropic
Type 3 Kinase linked receptors
Type 4 Nuclear receptors
32
How many classes of receptors are found on the cell surface?
three
33
What are the properties of receptors found on cell surface?
- Transmembrane-spanning segments composed of 20-25 hydrophobic amino acids
- Possess extracellular ligand binding domain
- many drugs act via the ligand binding domain
34
What are the properties of Nuclear receptors?
- DNA-linked
- regulate gene transcription
- ligand must be able to cross the plasma membrane
35
What are Ligand-gated ion channels?
Receptors are ion channels ionotropic receptors
- Involved in fast synaptic transmission (ms-ms)
- Endogenous ligands are fast/classical neurotransmitters eg. ACh
- Composed of 3-5 subunits (1 subunit =1 gene=1protein)
- Each subunit has 2-4 Transmembrane spanning domains (TMs)
- Complex arranged to form a central aqueous pore
- Agonist binding leads to channel opening (ms)
A very common drug target protein- ion channels found on excitable cells, neurones, muscle
Important in mediating the effect of a chemical mediator
Chemical mediator binds to a neurone/muscle causing an ion chancel to open, depolarisation and action potential
36
What do Ionotropic receptors do?
they form an ion channel pore
37
What are the three main classes of ligand-gated channels?
Nicotinic acetylcholine receptor
Glutamate receptor
P2X receptor
38
Name example of cys-loop receptors and the number to subunits and transmembrane domains they are
Nicotinic acetylcholine receptor
5-HT3 a ligand gated ion channels which respond to serotonin
GABAa ligand-gated ion which responds to gaba
Glycine receptor respond to amnio acid glycine on outside
Thus all cys-loop receptors are made up of 5 proteins (called subunits), each subunit has 4 transmembrane domains.
39
Give example of Glutamate receptors and how many subunits they have
AMPA
Kainate
NMDA
4 subunits
40
Give example of P2X receptors and how many subunits they have
ATP- gated
purinergic
3 subunits making up a channel
the simplest
41
Describe the structural aspects of nicotine receptors
- Each nicotinic receptor = HETEROMERIC assembly of five subunits (PENTAMERIC structure)
- Nicotinic subunits = gene products encoding: Alpha, Beta, Gamma, delta subunits (also epsilon)
- Receptors combine in distinct combinations
- TWO molecules of Ach (or nicotine) must bind to the ‘orthosteric site’ to activate the receptor (open the channel)
- These bind at INTERFACES between alpha subunits
42
What produces changes in excitability/potential of a cell?
Ions flowing through the open channels produce changes in the ‘excitability’ / ‘potential’ of the cell
Channel can be cation selective (sodium, potassium, calcium) OR anion selective (chloride)
43
When do Ligand-gated ion channels open?
When bound by an agonist
44
What happens when there's activation of Ionotropic Receptors for Excitatory Neurotransmitters? give exampe
- membrane depolarization
- Action potential firing
eg. nAChR
- Neuromuscular Junction and Brain
- Agonist = Nicotine
- Antagonist = tubocurarine
45
What happens when there's activation of Ionotropic Receptors for Inhibitory Neurotransmitters? give examples
- inhibit membrane depolarization
- reduce action potential firing
eg. GABAAR Brain
Agonist = GABA, Phenobarbitone*
Antagonist = Picrotoxin
46
What are G-protein coupled receptors?
- Largest family of cell surface receptors in mammalian genome
- All have protein Receptor polypeptide which spans the membrane 7 times (also known as 7TM-Rs, Serpentine)
- Not formed from multiple subunits*
47
Give examples of endogenous ligands
- hormones (eg. vasopressin, angiotensin),
- neuropeptides (eg. opioids)
- small molecule transmitters (eg. Ach, noradrenalin, Histamine), odorants, photons, pheromones, chemokines
- Opiates (morphine) bind to g-protein coupled receptor
48
What are Metabotropic receptors and give examples
they are a type of membrane receptor of eukaryotic cells that acts through a second messenger. It may be located at the surface of the cell or in vesicles.
- mAChRs, adrenoceptors, opioid receptors
- all neurotransmitters make use of GPCRs, but not all make use of Ligand-gated ion channels
- Specialized (distinct) functions according to signals they produce
49
What the g-protein coupled receptors do?
Regulate effectors proteins (enzymes or ion channels) via a G protein by generate ‘second messenger’
50
What do second messengers do?
Amplify the signal
51
What two ways could Signal transduction occur via GPCRs?
- Agonist binds to its receptor which allows receptor to interact with and activate g-protein
- Activated g-protein then goes on to control function of another protein
- could be regulating an ion channel
or. .
- Receptor through g protein may regulate activity of an enzyme to control production of second messengers
- Many examples where activation of receptor can lead to regulation of multiple effectors, g protein simultaneously many regulate ion channels AND enzymes
52
What's the function of heterotrimeric G-Proteins?
► Proteins that bind guanine nucleotides
GDP(rest) and GTP(activated)
GDP is bound to g-protein at rest- when receptor is activated and the g protein associates with receptor it causes a change in the structure of the g protein which makes it more attractive to GTP
GDP is EXCHANGED for GTP, when the g protein is activated by a receptor
53
What subunits are heterotrimeric G-Proteins composed of?
G-protein made of three subunits:
- Alpha
- Beta
- Gamma
Beta and gamma are always attached to each other, they never come apart
Both alpha and beta-gamma subunit allow receptor to ‘talk’ to the effector enzyme or ion channel
54
Describe G-protein activation
| use notes for a better description
► Agonist binding to the receptor → ‘activates’ the G protein
► Induces Exchange of GDP for GTP – in this sense the agonist bound receptor acts like a guanine nucleotide exchange factor (GEF) for the G protein
► Activated G protein dissociates into alpha and beta-gamma subunits → ‘activate’ or ‘inhibit’ effectors
► the activated G protein subunits dissociate from the receptor, leaving the receptor ‘free’ to activate more G proteins ie. one receptor can activate many G proteins and consequently affect the activity of many effector molecules → amplification of response
(use notes for a better description)
55
Describe Amplification of signals generated by GPCRs
- Way signalling work allows for a lot of amplification
- If the receptor is occupied by agonist its in a state to activate g proteins
- A single activated receptor can be activating multiple copies of g protein
- Each one of those active g proteins can regulate activity of multiple copies of effectors which they associate with
56
What are two ‘effectors’ that are regulated by GPCRs?
1. Enzymes which regulate levels of 2nd messengers
| 2. Ion channels-direct or indirect
57
Give examples GPCR's that regulate Enzymes which regulate levels of 2nd messengers
Types of Alpha subunits:
- Gs: has an alpha s subunit associated with it, it stimulates Adenyly cyclase to increase levels to cyclic AMP
- Gi: this subunit when activated inhibits adenyly cyclase and so decreases levels of cAMP
- Gt: responsible for transduction of light signalling
- Gq: regulates activity of enzyme phospholipase c by increasing it, which generates a different set of second messenagers. Increases IP3 which regulates release of calcium from inside cells
58
How are G-proteins named?
g-proteins are named by their alpha subunits, the little letters come from alpha subunits
59
Give examples of GPCR's that regulate Ion channels-directly or indirectly
eg. voltage-operated channels
‘opened’ or ‘closed’
increase or decrease of action potential firing
60
Describe GPCR regulation of an enzyme
cAMP is a 2nd messenger – small molecule regulates many proteins
- adenylyl cyclase (which is stimulated of inhibited by GCPR's) is a very important enzyme controlling the production of the second messenger cAMP from ATP. -- cAMP in turn regulates the activity of a kinase, protein kinase A which can go on to phosphorylate many different types of proteins with variable outcomes.
61
Receptor signalling via G-proteins can be either
Fast: PKA phosphorylating an enzyme to increase activity
Slow: PKA mediated activation of gene transcription (hours)
Note that PKA is a serine/threonine kinase regulated by cAMP
62
What else if duration of signalling determined by? what does it require?
What switches the signal off - Gi downregulates Adenylyl cyclase
Switching this off requires the phosphorylated protein to be dephosphorylated by a phosphatase.
Protein phosphatases terminate signalling initiated by pKa
63
What happens when Gq coupled receptors activate PLC? give an example
* Phospholipase C-b hydrolyses phosphatidylinsitol 4,5 bisphosphate (PIP2).
* This produces diacylglycerol (DAG) and inositol 1,4,5 triphosphate (IP3). Two more types of 2nd messengers
muscarininc receptors eg. M1 receptors, activated by parasympathetic postganglionic neurons and pilocarpine use this signaling cascade.
64
What happens during Signalling via IP3?
* IP3 diffuses to endoplasmic reticulum (ER).
* IP3 releases Ca2+ stored in ER into cytosol.
* Released Ca2+ triggers further signaling events.
65
How does Gq couple receptor regulate Termination of IP3?
- mediated increase in [Ca2+] i
- IP3 is dephosphorylated to IP2.
- IP3 is phosphorylated IP4.
- Ca2+ is pumped out of the cytosol.
66
How does Gq - protein couple receptor regulate diacylglycerol(DAG) activation of protein kinase C
* Ca2+ causes protein kinase C to move to the plasma membrane.
* In the plasma membrane, PKC is activated by DAG and bound Ca2+.
* Activated PKC phosphorylates many target proteins.
in this case through a calcium and diacylglycerol regulated kinase known as PKC.
67
Give example of Complex signalling
- Single GPCR can regulate multiple effectors
- Multiple GPCRs can couple to single effector
- Cross-talk between receptors
- Provides cell with ability to integrate multiple stimuli and thereby modify its response appropriately
- Intracellular coding of Extracellular changes
68
What can Enzyme-linked signalling be mediated by?
kinases or phosphatases
69
What are Acetylcholine and Noradrenaline?
transmitters of the peripheral nervous system
70
Name transmitters of the parasympathetic system and what organs are involved
Parasympathetic system- salivary glands- ACh (mus)- ACh (nic) - central nervous system
71
Name transmitters of the sympathetic system and what organs are involved
Sympathetic system-
Adrenal medulla- ACh (Nic)- CNS
Sweat glands - ACh (mus)- ACh (nic) - CNS
Blood vessels- NA - ACh (Nic) - CNS
72
Name transmitters of the somatic efferent system and what organs are involved
Somatic efferent system-
| skeletal muscle - ACh (Nic)- CNS
73
Nicotinic Receptor: Preganglionic neurons
* Always myelinated
* Always cholinergic fibres i.e. release ACh as their primary neurotransmitter
* ACh activates nicotinic ACh receptors on the postsynaptic cell, ionotropic ligand gated ion channels
74
Nicotinic subunit composition in ANS
What is the composition of post ganglionic nicotinic receptor?
What the composition of skeletal muscle nicotinic receptor?
What's a property of nicotinic receptors?
There are different types of nicotinic Ach receptors expressed in different types of neurons and different tissues
Post ganglionic nicotinic receptors have a subunit composition, composed of two alpha 3 subunits and three beta 4 subunits
Skeletal muscle nicotinic receptors have a different subunit composition, two alpha 1 subunit, beta, delta and epsilon subunit
Both nicotinic receptors, ligand-gated ion channels, activated by Acetylcholine- but nicotinic receptors can be selectively targeted over another nicotinic receptor
75
Drugs may act indirectly to enhance cholinergic transmission
► Inhibit Cholinesterase*
Acetylcholinesterase (AChE) CSF, synaptic cleft at cholinergic synapses & cholinergic nerve terminals
Anticholinesterase drugs classified by duration of action
► Short acting (reversible binding) edrophonium (use limited to diagnosis of myasthenia gravis)
► Medium (slow hydrolysis) neostigmine & pyridostigmine (may be used in myasthenia gravis as don’t cross BBB)
physostigmine (crosses BBB – used topically for glaucoma)
Long (irreversible) nerve gas,
organophosphates, pesticides
(cross BBB)
76
What are the two muscarinic receptors and which G-proteins do they work with?
```
5 subtypes (3 well characterised)
Postsynaptic in Parasympathetic ganglion neurones and sweat glands
```
M1 receptors found on smooth muscle cell and endo-exocrine gland couple to Gq protein
Gq regulate active of phospholipase c to generate two intracellular messengers
M2 receptors has a presence for association with Gi heterotrimeric G protein, well known to regulate activity of ion channels, ie inhibit voltage gated calcium ion channels on a pre- synaptic location to decrease neurotransmitter release
77
What receptor activates PLC (phospholipase C)?
M1 mAChR - Gq coupled receptors
* Phospholipase C-b hydrolyses phosphatidylinsitol 4,5 bisphosphate (PIP2).
* This produces diacylglycerol (DAG) and inositol 1,4,5 triphosphate (IP3). Two more types of 2nd messengers
78
Signalling via IP3
* IP3 diffuses to endoplasmic reticulum (ER).
* IP3 releases Ca2+ stored in ER into cytosol.
* Released Ca2+ triggers further signaling events
* secretion from glands, contraction of smooth muscle.
79
Importance of calcium- what does it's release promote?
The rise of calcium in smooth muscle contributes to the contraction of the muscle
In other cell types calcium may regulate other effectors, including ion channels
The calcium released from store in endocrine glands promotes secretion of hormones
80
Gq: Termination of IP3 -mediated increase in [Ca2+] i
1. IP3 is dephosphorylated to IP2.
2. IP3 is phosphorylated IP4.
Ca2+ is pumped out of the cytosol
termination of action of signalling by these receptors involves breakdown of second messenger IP3 and removal of calcium from the cytosol back into the store where is came from- this terminated the signalling
81
Muscarinic Receptor Regulation of Potassium
Channels in the Heart
Parasympathetic regulation of the heart
Parasympathetic nervous system: rest and relax
Heart rate slows down
Post ganglionic parasympathetic neurons use ACh muscarinic receptors
When muscarinic receptors in the heart are activated and G proteins, the heart rate slows down by the beta gamma subunits which dissociate from the active receptor, binding to a potassium channel which is found in pacemaker cells opening potassium channel, where the cells hyperpolarise, so decrease action potential firing and the heart rate slows down.
GPCRs effector functions mediated by alpha subunits and beta gamma subunits depending on the tissue there will have different consequences
82
Cholinergic agonist at mAChR- name one and its effects
```
Muscarine -(Amanita muscaria )
Eating over 1 gram can cause nausea
Other effects, depend on dosage inc.
decrease in blood pressure
↑saliva, ↑ tearflow ↑ sweating
abdominal pain
Death from cardiac + respiratory failure
```
Pilocarpine eye drops- used to treat glaucoma
83
Cholinergic antagonists- name an antagonist against mAChR and its effects
```
Atropine
EFFECTS
Inhibition of secretion
salivary, lacrimal, bronchial, sweat
Smooth muscle relaxant
bronchial, biliary, urinary tract
Pupillary dilation
(modest ↑ in HR)
(↓ GI motility)
CNS - agitation and disorientation
↑ body temperature
```
Effects may be used to reverse poisoning by anticholinesterases
84
What does the Sympathetic pre-ganglionic neurons synapse with?
What's the neurotransmitter in the sympathetic nervous system?
adrenal medulla
Adrenaline is one of the neurotransmitters in the sympathetic nervous system
85
Adrenergic receptor subtypes are classified by
pharmacology, G protein coupling and effector systems they regulate
86
Adrenergic regulation in cardiovascular system
* b1 adrenoceptors (Gs coupled) positive ionotropic and chronotropic effects in the heart
* a2 adrenoceptors, (Gi coupled) presynaptic inhibition of NA release
Gs = asbg
as is stimulatory
Gi = aibg
ai is inhibitory
87
Clinical uses of adrenoceptor agonists
► Cardiovascular system
Cardiac Arrest Adrenaline
Anaphylaxis Adrenaline
Hypertension Clonidine (a2 agonist)
► Respiratory system
Broncodilator Salbutamol (b2 selective)
Nasal decongestant Ephedrine (b agonist & indirectly acting sympathomimetic drug ie. causes NA release)
88
Clinical Uses Adrenoceptor Antagonists
► Hypertension prazosin (a1 selective)
► Heart failure carvedilol (a & b)
► Anxiety (somatic symptoms) propranolol (b1 & b2)
89
Unwanted effects for adrenoreceptor antagonists
```
• Bronchoconstriction*
“beta blockers” avoided in Asthma Patients
• Cardiac depression (elderly)
• Bradycardia
• Fatigue
• Cold extremities
```
90
Cross-talk and other neurotransmitters in the ANS
| contribute to what? name examples
maintenance of homeostasis – and drug effects!
• Many other Non-Adrenergic, Non-Cholinergic “NANC” neurotransmitters are present inc.
• substance P (GPCR)
• ATP (ligand gated P2X and P2Y GPCRs)
• VIP (GPCR)
• nitric oxide (diffusable messenger acts on an enzyme)
91
Presynaptic inhibitory GPCRs help prevent what?
| What must we be careful of?
prevent overstimulation of ANS function
Must not forget possible impact of interfering with these when using drugs eg. clonidine
92
Name ACh receptors and NA receptors
Where are they neurotransmitter?
What do they regulate?
o ACh (nAChR and mAChR) and NA (alpha and beta receptors) are the principal neurotransmitters of the Autonomic Nervous System, regulate cardiovascular system
93
Scaffolding proteins:
- Scaffold proteins play an important role in coordinating signal transduction cascades.
- Controlling protein-protein interactions- bind adaptors, restrict/specialise type of signalling, strengthens interactions, avoids signalling chaos
