Membranes and Receptors Flashcards
(360 cards)
1
Q
List 5 functions of biological membranes
A
- Continuous, highly selective permeability barrier
- Control of the enclosed chemical environment
- Communication
- Recognition - signalling molecules
- adhesion proteins
- immune surveillance - Signal generation in response to stimuli.
2
Q
What is the general dry eight membrane compostion?
A
40% lipid
60% protein
1- 10% carb
3
Q
What does amphipathic mean?
A
They contain both hydrophilic and a hydrophobic moiety
4
Q
What is the composition of a phospholipid molecule?
A
Fatty acid chain (all same length- C14-24)
Glycerol
Phosphate
Polar head group (eg choline, amines, amino acids, sugars…)
5
Q
What causes the kink in the fatty acid tail?
A
Cis double bond
6
Q
What’re the 2 types of glycolipids?
A
Cerebrosides- sugar monomer head group
Gangliosides- oligosaccharide (sugar multimers) head group
7
Q
What are the 4 types of motion occurring by the phospholipids in the membrane?
A
Flexing
Rotation
Lateral diffusion
Flip flop
8
Q
What influence does the cis double bond have in the unsaturated hydrocarbon chains of the membrane?
A
Reduces phospholipid packing
9
Q
What are the 3 main components of a cholesterol molecule?
A
Polar head group
Rigid planar steroid ring structure
Non-polar hydrocarbon tailor.
10
Q
What does the rigid steroid ring do in cholesterol?
A
Restrict motion
11
Q
What is the evidence for protein membranes?
A
Functional:
- facilitated diffusion
- ion gradients
- specificity of cell responses
Biochemical:
- membrane fractionation and gel electrophoresis
- freeze fracture
12
Q
Which fracture face contains the cytosol?
A
P face
13
Q
What are the 3 modes of motion of proteins in the membrane?
A
Rotation
Conformational change
Lateral diffusion
NO FLIP FLOP
14
Q
How can mobility of proteins be restricted?
A
Aggregation
Tethering
Interaction with other cells
15
Q
What causes restraints on protein mobility in the membrane?
A
Lipid mediated effects- separate out into the fluid phase or cholesterol deficient regions
Membrane and peripheral protein associations
16
Q
What is a peripheral membrane protein?
A
Bound to the surface by electrostatic forces and hydrogen bond interactions. Can be removed by changes in the pH or ionic strength
17
Q
What is an integral protein?
A
A protein in the membrane that interacts extensively with hydrophobic domains of the lipid bilayer
18
Q
How are integral proteins removed?
A
By agents that compete for non-polar interactions e.g. Detergents and organic solvents
19
Q
What is a transmembrane polypeptide.
A
A protein that goes from one side of a membrane through to the other side. Usually contains hydrophobic R groups and exists as a helix
20
Q
What is membrane protein topology?
A
The location of the N-terminus (inner or outer side of the membrane)
21
Q
Describe secretory protein synthesis.
9 points
A
- Free ribosomes initiate protein synthesis from mRNA molecule.
- Hydrophobic N-terminal signal sequence is produced.
- Signal sequence is recognised and bound to by the SRP
- Protein synthesis stops
- GTP-bound SRP directs the ribosome synthesising the protein to SRP receptors on the cytosolic face of the ER.
- SRP dissociation
- Protein synthesis continues and the newly formed polypeptide is fed into the ER via a pore in the membrane (peptide translocation complex)
- Signal sequence is removed by a signal peptidase once the entire protein has been synthesised.
- The ribosome dissociates and is recycled.
22
Q
What additional step is there in membrane protein synthesis compared to secretory protein synthesis?
A
stop transfer signal.
When the membrane protein is being translated into the ER lumen, the ribosome comes across a highly hydrophobic stop transfer signal.
23
Q
What does a hydropathy plot show?
A
How many transmembrane regions a protein has.
24
Q
What is membrane asymmetry important?
A
For function. Position of the recognition site is important.
25
How does the cis formation of the unsaturated fatty acid side chains affect fluidity?
introduces a kink in the chain that reduces phospholipid packaging which increases membrane fluidity.
26
How does cholesterol stabilise the plasma membrane?
By hydrogen bonds to he fatty acid chains. This abolishes the endothermic phase transition of phospholipid bilayers.
27
How does cholesterol increase membrane fluidity?
Reduces phospholipid packing. However it also reduces phospholipid chain motion, decreasing membrane fluidity.
28
Why is the plasma membrane referred to as a fluid mosaic model?
fluid- it is not solid due to the hydrophobic integral components such as lipids and membrane proteins that move laterally throughout the membrane.
Mosaic- Made up of many different parts...
29
What effects lateral diffusion of proteins through the the membrane?
```
Size
Protein aggregation
Association
Lipid mediated effects
Proteins tend to separate out into fluid phase or cholesterol poor regions.
```
30
What composes the erythrocyte cytoskeleton/
Spectrin actin molecules attached to the membrane by adapter proteins Ankyrin and Glycophorin.
31
How does the cytoskeleton effect mobility of membrane proteins?
restricts lateral mobility by attachment to integral membrane proteins
32
What happens to erythrocytes without the cytoskeleton?
More spherical and lysed by shearing forces in capillary beds and cleared by the spleen
33
What is hereditary spherocytosis?
Spectrin levels depleted (by 40-50% in dominant form) resulting in rounding up and increased lysis of cells reducing RBC lifespan. This leads to haemolytic anaemia as the bone marrow cannot compensate.
34
What is hereditary elliptocytosis?
Spectrin molecules are unable to from heterotetramers resulting in fragile elliptoid cells which leads to haemolytic anaemias
35
Describe passive diffusion.
Dependent on permeability and concentration gradient with the rate of passive transport increasing linearly with increasing concentration gradient.
36
Describe facilitated diffusion.
Diffusion across the membrane with the help of a specific protein in the bilayer eg carrier protein or protein channels.
37
Describe active transport.
Transport of ions or molecules against an unfavourable concentration gradient and/or electrical gradient, requiring energy form ATP.
38
What are co-transporters?
Membrane transporters that transport more than one molecule.
39
What is a uniport?
Single transport molecule working in one direction
40
What is a symport?
A unit that transports 2 molecules in the same direction
41
What is an antiport?
A unit that transports 2 molecules in opposite directions.
42
What binds to the alpha subunit in the Na/K pump?
K
Na
ATP
Oubain
43
What binds to the beta subunit of the Na/K pump?
Glycoprotein which directs the pump to the surface.
44
What does oubain do?
Inhibits Na, K and ATP binding
45
What type of transport does Na/K ATPase involve?
Antiport- 2K in, 3Na out
46
What are the results of transport from the Na/K pump?
Forms NA and K gradient necessary for electrical excitability which drives secondary active transport
47
How does Na and K regulation effect the cell?
```
Control of pH
Regulation of cell volume
Regulation of Ca2+ conc
Absorption of Na in epithelia
Nutrition uptake
```
48
What does PMCA do?
Regulates Ca using ATP.
Expels Ca for the cell in exchange for H ions making the cell more alkaline.
High affinity, low capacity
49
What does SERCA do?
Transports Ca into the SR in exchange for H.
High affinity, low capacity
Removes residual Ca
50
What does NCX exchange?
1 Ca out for 3 Na in
51
How does NCX work?
Uses the Na concentration gradient set up by Na/K ATPase antiport
Membrane potential dependent.
Low affinity high capacity
52
How does NCX change in activity in ischaemia?
Sodium pump is inhibited due to ATP depletion
| Na accumulates in the cell so NCX reverses
53
Name the main acid extruders.
NHE - Na/H Exchanger
| NBC - Sodium Bicarbonate Co-Transporter
54
How does NHE work?
Exchanges Na for intracellular H raising the inrtracellular pH
55
What activate NHE?
growth factors
56
What inhibits NHE?
amiloride
57
Other than being an acid extruder, what other function does NHE have?
Regulates cell volume
58
What other function does NBC have?
Involved in regulating cell volume
59
What is the bodies main base extruder?
AE - Anion Exchanger. Cl/HCO3 exchanger
| Removes base from the cell
60
What is another function of the base extruder?
cell volume regulation
61
How does ion transport regulate cell volume?
Water follows the ions, causing cell shrinkage or swelling.
62
Why is the Na/K pump important in the proximal tubule?
Keeps intracellular Na conc low so NHE can pump H ions into the proximal tubule lumen.
63
What is the importance of H in the proximal tubule?
"Picks up" bicarbonate and brings it back into the cell
64
What is the aim of anti-hypertensive therapy?
Reduce the reuptake of Na and other molecules so less water is reabsorbed via osmosis
With less water being absorbed, blood volume and therefore pressure will fall
65
What does aquaporin do?
Allows water to readily cross the membrane of the proximal tubule. It's inclusion in the membrane is stimulated by anti-diuretic hormone
66
What are loop diuretics?
Block Na reuptake in the thick ascending limb of the prox. convoluted tubule
67
What works in the prox and distal convoluted tubules to prevent Na uptake?
Amiloride
68
What does aldosterone do in the kidneys?
Works to up-regulate sodium transporters in the prox and distal convoluted tubule
69
What is used to treat abnormally high levels of aldosterone?
Spironolactone (Glucocorticoid receptor antagonist)
70
How does Na transport by the Na/K pump afftect Cl?
Allows the symport of 2Cl into the cell with Na and K
71
What causes cystic fibrosis?
Faulty CFTR protein leads to accumulation of C ini the cell, water moves into the cell by osmosis leading to thick, viscous mucous in the lumen.
72
How does diarrhoea occur?
CFTR is overly active and phosphorylated by Protein Kinase A
Cl is excessively transported into the lumen
Water follows, giving the symptoms of diarrhoea
73
What is a membrane potential?
The electrical potenetial difference across a cell membrane.
74
What is the resting membrane potential?
Potential inside the cell relative to teh extracellular solution
75
How can a membrane potential be measured?
Using a micorelectrode which penetrates the cell membrane and is filled with conducting solution (KCL)
76
What is the range of animal cell membrane potentials at rest?
-20 to -90mV
77
What part of the body has the largest resting potential?
Cardiac and skeletal muscle -80 to -90mV
78
What is the resting potential of nerve cells?
-50 to -75mV
79
What determines what the membrane selectively permeable to ions?
Protein channels.
80
How is the resting potential of the cell set up?
The selectivity of ion channels and the type of channels that are open make the whole cell membrane selectively permeable to ions which determine the potential
81
What ion dominates the membrane ionic permeability at rest?
potassium
82
How does K set up the membrane potential?
When chemical and electrical gradients of K are equal and opposite, there is no net movement of K but there will be a negative membrane potential.
83
What is the equilibrium potential for an ion?
the membrane potential at which there is no net movement of the ion across the membrane
84
What is used to calculate the equilibrium potential?
The Nernst equation
85
Define depolarisation
The membrane potential decreases in size (but not necessarily enough to form an action potential - may be v small change) Cell interior becomes less negative
86
Define hyperpolarisation
Membrane potential increases in size - falls below resting. Cell interior becomes more negative
87
Where do synaptic connections occur?
between nerve, muscle, sensory cells and glands
88
What type of synaptic transmission are there?
Fast and Slow
89
What is fast synaptic transmission?
The receptor protein is also an ion channel. Binding of transmitter causes the channel to open
90
What is slow synaptic transmission?
The receptor protein and ion channel are separate. May be linked by a G protein or intracellular messengers
91
What do excitatory transmitters do?
Open ligand-gated channels causing membrane depolarisation
92
What is an EPSP?
Excitatory Post Synaptic Potential
Has a longer time course than an AP
Graded with the amount of transmitter (acetylcholine, glutamate)
93
What do inhibitory transmitters do?
Open ligand-gated channels causing hyperpolarisation - permeable to K or Cl
94
What is an IPSP?
Inhibitory Post Synaptic Potential
| Transmitters include Glycine, gama-aminobutyric acid (GABA)
95
What is an action potential?
Change in voltage across the membrane
Depends on ionic gradient and relative permeability of the membrane
Only occurs if a threshold value is reached - All or nothing
Propagated without loss of amplitude
96
On investigation of the mechanism of action potential generation, what does voltage-clamping do?
Controls the membrane potential so that the ionic currents can be measured
97
What does path clamping do?
Enables currents flowing rough individual ion channels to be measured
98
What is the mechanism of the upstroke of the AP?
Positive feedback of the depolarisation of the membrane on opening of the Na channels
99
How is the cell repolarized?
Further depolarisation causes the Na channels to shut and the K channels to open causing repolarisation
100
What is an ARP?
Nearly all Na channels are in the activated state
| Absolute Refractory Period
101
What is the RRP?
Na channels are recovering from inactivation, the excitability returns to towards normal as the number of channels in the inactivated state decreases
102
What is accommodation?
The longer the stimulus, the larger the depolarisation necessary to initiate an action potential - the threshold becomes more positive
103
In a voltage gated Na channel, which subunit (?) is the voltage sensor?
4 - has a positive amino acid residue
104
What does the P or H5 region contribute to?
Pore selectivity
105
How do many local anaesthetics work?
By blocking Na channels
106
In what order do local anaesthetics block axons?
1. Small myelinated
2. Un-myelinated
3. Large myelinated
107
Name 2 diseases that affect conduction of an action potential in the CNS
Multiple Sclerosis - All CNS nerves
| Devics disease - Optic and Spinal nerve only
108
Name 2 diseases that affect conduction of an action potential in the PNS
Landry-Guillin-Barre syndrome
| Charcot-Marie-Tooth disease
109
What is capacitance?
The ability to store charge - a property of the lipid bilayer
110
What does membrane dependence rely on?
The number of ion channels open - lower resistance, more channels open
111
What does the spread of local current depend on?
Capacitance and membrane resistance
112
What causes the rapid upstroke of the AP?
Influx of Na
113
What increases the rate of the spread of the action potential?
High resistance and low capacitance
114
What happens if the myelin sheath is damaged?
Saltatory conduction is inhibited
115
How does an action potential cause Ca channels to open?
The depolarisation of the membrane causes the voltage gated channels to open.
116
How do Ca channels differ?
Affected by different blockers
117
How is the difference in Ca channels beneficial?
Localised effect of blockers - Different channels have different primary locations
118
How is the high density of Ca channels at nerve endings beneficial?
Provides a large enough influx of Ca to trigger Ach release
119
How does Ca cause the release of Ach?
Binds to Synaptotagmin, leading to the formation of the Snare Complex and Ach release
120
What does binding of Ach with nicotinic receptors on the post-junctional membrane produce?
An end-plate potential
121
What does this depolarisation of the end plate do?
Raises the muscle above threshold so an action potential is evoked in the muscle membrane
122
Name 2 types of blockers that work on nicotinic receptors
competitive
| depolarising
123
How do competitive nicotinic blockers work?
Bind at the molecular recognition site for Ach
| eg. Tubocurarine
124
How do depolarising nicotinic blockers work?
Cause a maintained depolarisation at the post-junctional membrane. Adjacent Na channels will not be activated due to accommodation
eg. Succinylcholine
125
What is Myasthenia Gravis?
Autoimmune disease targeting nicotinic receptors
126
What are the symptoms of Myasthenia Gravis?
Drooping eyelids
Weakness
fatigue
127
How is Myasthenia Gravis treated?
Ach-esterase inhibitors. Increase the amount of time Ach is in the synaptic cleft
128
Why is it important to regulate intracellular Ca?
Many cellular processes are Ca sensitive and it cannot be metabolised.
129
How is Ca regulated?
Largely by moving Ca into and out of the cytoplasm
130
What are the advantages and disadvantages of the large Ca gradient?
Advantages- Changes in intracellular Ca occur rapidly with little movement
Disadvantages- Ca overload leads to loss of regulation and cell death
131
What does the Ca gradient rely on?
Relative impermeability of the membrane
Ability to expel Ca (using Ca ATPase and Na/Ca exchanger
Ca Buffers
Intracellular Ca stores (rapidly and non rapidly releasable)
132
What is the relative impermeability of the membrane dependent on?
open/closed state of the ion channels
133
How do Ca buffers effect Ca gradient?
Limit diffusion through ATP and Ca binding proteins
134
Name 4 proteins which may act as Ca buffers
Parvalbumin
Calreticulin
Calbindin
Calsequestin
135
What does diffusion of Ca depend on with regards to buffers?
THe conc of binding molecule and it's level of saturation
136
What is the basal concentration of Ca in most cells?
100nm
137
What level can the Ca concentration rise to when it is being used to regulate cell activity?
1 micrometer
138
How is the concentration of Ca altered?
Influx across the plasma membrane
Release from "rapidly releasable" stores
Release from "non-rapidly releasable" stores
139
How is the permeability of the membrane to Ca altered?
Voltage-Gated Calcium Channels (VGCC)
| Receptor Operated Calcium Channels (Ionotropic receptors - Ligand/agonist binds to the channel)
140
Where are calcium stores in the cell?
Sarco/Endoplasmic reticulum, set up by SERCA protein. Moved in using ATP energy and binds to proteins
141
What might cause Ca to be released from the sarcoendoplasmic reticulum?
ligand binding to G-Protein coupled receptor on the cell membrane, activating its alpha subunit. This then binds to the membrane phosphlipid PIP2 releasing IP3 which in turn binds to its receptor on the sarcoendoplasmic reticulum , triggering the release of Ca down its conc grad.
Ca induced release - Ca binds to the Ryanodine receptor on the S/ER triggering Ca release
142
Where is CICR physiologically important?
Cardiac myocyte
143
When/why is Ca taken up into the mitochondria?
When the Ca conc is high as a protective mechanism. They aid in buffering, regulating signalling and stimulation of ATP production
144
How do mitochondria take up Ca?
Via a uniport driven using respiration
145
What is required to return Ca to it's basal level?
Terminationi of signal
Ca removal
Ca store refilling
146
What refills the Ca stores?
Recycled cyytosolic Ca
| Ca in the mitochondria is used to replenish SR stores
147
How is mitochondrial Ca transferred to SR stores?
Via store-operated Ca channel (SOC)
148
Define receptor
A molecule that recognises specifically a second molecule or family of molecules and in response to binding, brings about the regulation of a cellular process. SILENT AT REST
149
How are receptors classified?
By their specificity to a physiological signalling molecule. They are often divided further on the basis of their affinity to a series of antagonists
150
What are some roles of receptors?
Signalling
Neurotransmission
Cellular delivery
151
What is the difference between a receptor and acceptor?
Receptors are silent at rest and require a ligand to be activated. Acceptors operate in the absence of ligands
152
Define a ligand
A molecule that binds specifically to a receptor site
153
What is an agonist?
A ligand that activates a receptor on binding
154
What is an antagonist?
A ligand that binds to a receptor without causing activation and blocks the receptor
155
How do hydrophobic molecules travel in the blood and cross membrane barriers?
They may require carrier proteins which are hydrophilic to travel in the blood but can diffuse straight across the membrane lipid bilayer (if small enough)
156
Where are receptor sites for small, hydrophobic molecules located?
Inside the cell
157
Where are receptor sited for hydrophilic molecules?
On the cell surface
158
How do hydrophilic molecules bring about changes inside the cell?
Signal transduction. Extracellular receptor at the cell surface transmits the signal into the cell
159
How do membrane bound receptors cause signal transduction?
Integral ion channels
Integral enzyme activity
Coupling to effectors through transducing proteins
160
How does agonist binding to a ligand-gated ion channel cause it to open?
Brings about conformational change
161
What are classical ligand gated ion channels?
Share the similar pentameric subunit structures with four transmembrane domains
162
What happens to Tyrosine Kinase linked receptors upon ligand binding?
Autophosphorylate and are recognised either by transducing proteins or directly by enzymes containing phosphtyrosine recognition sites
163
How is the effector enzyme, Tyrosine Kinase activated?
On association with the receptor, allosterically activated by tyrosine phosphorylation by the receptor kinase. This transduces the message into an intracellular chemical event
164
What are G protein coupled receptors?
7 transmembrane domain receptors couple to effector molecules via a transducing molecule, a GTP binding regulatory protein
165
What are the effectors of G protein coupled receptors?
Enzymes or ion channels
166
What is integrated signalling?
often G protein coupled receptors will act simultaneously to inhibit and stimulate the effector. The 2 inputs combine to produce a measured effect
167
What are intracellular receptors bound to in their resting state?
heat shock or chaperone proteins
168
How do intracellular receptors work?
On activation, dissociate from the stabilising protein and translocate to the nucleus where it binds to control regions in DNA, regulating gene expression
169
Do intracellular or extracellular receptors work faster? Why?
Exracellular. The action of Intracellular receptors is dependent on transcription and translation
170
Why is amplification often necessary in extracellular signalling?
Concentration of extracellular signalling molecules is very low
171
What is amplification?
The binding of a chemical signal molecule to a single receptor can cause the modification of many substrate molecules
172
What does noradrenaline bind to to cause an increase in heart rate?
Beta-1- adrenoreceptors
173
WHat binds to M2 muscarinic receptors to cause slowing of the heart rate?
Acetylcholine
174
Where does phagocytosis occur?
Specialised cells - macrophages and neutrophils
175
Describe phagocytosis
1. In response to binding of a particle to receptors in the plasma membrane, the cell extends pseudopods that permit furthur receptor interactions
2. Membrnae invagination/particle internalisation via a membrane zippering mechanism
3. Internalised phagosomes fuse with lysosomes to form phagolysosomes in which the particulate material is degraded
176
What does phagocytosis do?
Clears damaged cellular material and invading microorganisms
177
What is pinocytosis?
The invagination of the plasma membrane to form a lipid vesicle
178
What does pinocytosis do?
uptake of impermeable extracellular solutes
179
What are the 2 forms of pinocytosis?
Fluid phase
| Receptor mediated endocytosis (RME)
180
What is receptor mediated endocytosis?
Specific binding of molecules to cell surface receptors permits the selective uptake of substances into the cell
181
Where are LDL's made?
Liver
182
What are LDLs made of?
core of cholesterol molecules esterified to fatty acid, surrounded by a lipid monolayer containing phospholipids, cholesterol and a single protein species, Apoprotein B
183
What do LDL receptors recognise?
Apoprotein B
184
Where are LDL receptors?
Cell surfaces, localised in clusters over Clathrin Coated Pits that cover approx 2% of the cells surface (spontaneous formation of pits and clathrin cages)
185
What happens when LDL binds to cell receptors?
Pits invaginate and form coated vesicles. Vesicles are uncoated in a process that requires ATP and fuse with smooth vesicles, endosomes
186
What is the pH of the endosome and how is it maintained?
5.5-6, lower than the cytoplasm, maintained by ATP dependent proton pump
187
Why does the endosome have a lower pH?
LDL receptor has a lower affinity for the LDL particle so dissociate
188
What is the endosome that dissociates the ligands and receptor known as?
Compartment for the Uncoupling of Receptor and Ligand (CURL)
189
What happens to the LDL receptor once dissociated from the particle?
sequestered to a domain within the endosome membrane which buds off as a vesicle and recycles the LDL-receptor to the plasma membrane
190
What happens to the LDL in the endosomes?
Endosomes fuse with lysosomes and the cholesterol is hydrolysed from the esters and released into the cell
191
What are the 3 mutations affecting the LDL receptor?
Non-functioning receptor - mutation to the binding site
No interaction of the receptor and clathrin coat so LDL receptors distributed over the entire cell surface
Receptor deficiency - expression of receptors prevented
192
How is transferrin formed in the circulation?
Binding of 2 Fe3+ ion to apoptransferrin
193
What happens when transferrin reaches the acidic endosome?
Fe ions are released but apoptransferrin remains associated to the transferrin receptor
194
What happens to the apoptransferrin after Fe is dissociated?
Sorted in the curl for recycling back to the membrane where apoptransferrin dissociates from the receptor again.
195
When do insulin receptors congregate over clathrin coated pits?
When insulin is bound to it - induces a conformational change in the receptor which allows i to be recognised by the pit
196
What happens to insulin in the endosome?
It remains bound to the receptor and is targeted to the lysosomes for degradation
197
How is a cell desensitised to a presence of high circulating insulin concentrations?
Lack of receptors associated with the cell membrane - only associates when insulin is bound to the receptor
198
What is Transcytosis?
Ligands remain bound to their receptors and are transported across the cell
199
How is immunoglobulin A from the circulation transported to bile in the liver?
Transcytosis. During transport the receptor is cleaved, resulting in the release of immunoglobulin with a bound secretoroy component derived from the receptor
200
How do membrane enveloped viruses and toxins enter cell?
Exploit endocytic pathways after adventitious binding to receptors in the plasma membrane
201
What happens to the membrane enveloped virus once in the endosome?
Acidic pH allows the viral membrane to fuse with the endosomal membrane releasing the viral RNA into the cell where it can be translated and replicated by the host cell's machinery to form new viral particles
202
What are G-proteins?
guanine nucleotide binding proteins
203
What is a G-protein coupled receptor?
A family of receptors that act by altering the activity of effectors via the activation of a G-protein
204
How are G-proteins hetertrimeric?
They have 3 distinct subunits, alpha, beta and gama. B and g are bound tightly together and function as a single unit
205
Where is the guanine nucleotide binding sit on the G-protein?
Alpha subunit
206
What does the guanine nucleotide binding site do?
Binds to the GTP and slowly hydrolyses it to GDP (GTPase activity)
207
Where is the G-p under basal conditions?
the inner face of the plasma membrane in it's heterotrimeric form (mostly) and GDP bound to the alpha subunit
208
When is the GDP released from the alpha subunit?
When an agonist binds to the GPCR and protein protein interactions occur between the heterotrimeric G-p and the receptor. GTP binds in it's place
209
What happens to the G-p when GTP binds?
Affinity of receptor for Alpha GTP and the BG subunit decreases. They are released and are each able to interact with effectors
210
What terminates the effector interaction of G-p subunits?
intrinsic GTP activity of the Alpha subunit hydrolysing GTP->GDP.
Affinity of A sub and BG sub increases and the ABG heterotrimer is reformed.
211
Describe the mechanism of action of a G-protein.
- Agonist binds to receptor
- Protein-protein interactions releases GDP, binds GTP
- Alpha-GTP and BG released and interact with effectors
- GTP hydrolysed to GTP
- Alpha-GDP and BG reform heterotrimer
212
What are 4 G-proteins?
Gs
Gi
Gq
Gt
213
What does Gs do?
Stimulates adenylyl cyclase
214
What signals Gs?
Adrenaline/noradrenaline
215
What receptor does Gs act on?
B-adrenoreceptor
216
What physiological response does Gs produce?
Glycogenolysis
| Lipolysis
217
What does Gi do?
Inhibits the action of adenylyl cyclase
| Stimulates K channels
218
What signals Gi?
Acetylcholine
219
What receptor does Gi act on?
M2-Muscarinic
220
What physiological response does Gi evoke?
slowing of cardiac pacemaker
221
WHat does Gq do?
Stimulate phosphlipase C
222
What signals Gq?
Acetylcholine
223
What type of receptors do Gq act on?
M3-Muscarinic
224
What physiological response does Gq evoke?
Smooth muscle contraction
225
What does Gt do?
Stimulates cyclic GMP phosphodiesterase
226
What signals Gt?
Light
227
What receptor does Gt act on?
Rhodopsin
228
What physiological response does Gt evoke?
Visual excitation
229
What receptors does Gs utilise?
adrenergic B1 and B2
230
What receptors does Gi utilise?
adrenergic A2 and cholinergic M2
231
What receptors does Gq utilise?
adrenergic A1 and cholinergic M1 and M3
232
How many G-protein combinations can the human genome encode?
1,000 possible combinations
20 alpha
5 beta
12+ gama
233
How many receptor types are there for G-proteins?
at least 800
234
How many enzyme/ion channel effectors can each G-p receptor activate?
10+
235
What does cholera toxin (CTx) do?
Inactivates G-alpha GTPase. G-alpha is permanently activated
236
What does pertussus toxin (PTx) do?
Interferes with the GDP->GTP exchange on G-alpha leading to its irreversible inactivation
237
Name 3 diseases caused by mutations in GPCRs.
Retinitis pigmentosa
Nephrogenic Diabetes Insipidus
Familial Male Precocious Puberty
238
What causes Retinitis pigmentosa?
Loss of function mutation to rhodopsin
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What causes Nephrogenic Diabetes Insipidus?
Loss of function mutation to V2 vasopressin receptor
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What causes Familial Male Precosious Puberty?
Gain of function he Luiteinising Hormone receptor
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How does cyclic AMP exert the majority of it's actions?
Through cyclic AMP-dependent Protein kinase (PKA)
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What activity does activation of cyclic AMP receptors cause in the body?
Glycogenolysis and gluconeogenesis
Lipolysis
Relaxation of many smooth muscles
Positive inotrophic and chronotrophic effectrs on the heart
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What does phospholipase C do?
Hydrolyses membrane phospholipids to to IP3
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What does IP3 do?
interacts with receptors on ER membrane to activate the release of Ca from the lumen and enter the cytoplasm
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What does cyclic GMP phosphodiesterase do?
found in the photoreceptive cells in the retina
| regulates the breakdown of the secondary messenger cyclic GMP phosphodiester
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How does dissociation of a G-p and receptor occur?
Once they have productively interacted, the binding of the agonist is weakened - dissociation occurs
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How is further interaction with other G-p prevented when the receptor is activated? (receptor desensitisation)
When activated, the receptor is susceptible to a variety of protein kinases that phosphorylate the receptor and prevent it activating further G-ps
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How is the active lifetime of GTP limited?
Cellular factors stimulate the intrinsic GTPase activity of the G-alpha subunit
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What opposes the effect of second messengers/protein kinases downstream?
Enzymatic cascades activated
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What chemical in the body affects the rate at which the SAN fires an action potential?
Ach release in the parasympathetic nerves acting on M2 muscarinic cholinoreceptors
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How does Ach affect heart rate?
acts on M2 muscarinic cholinoreceptors to increase the open probability of K channels via Gi -> hyperpolarisation, slowing the intrinsic firing rate.
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What type of chronotropic effect does Ach have on the heart?
Negative chronotropic effect
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How is inotropy regulated in the heart?
Sympathetic innervation of the ventricles and/or adrenaline influence the force of contraction
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How does the activation of Beta-Adrenoreceptors cause positive inotropic effects?
Increased probability of open VOCCs, directly and indirectly by the activation of Gs. Influx of Ca causes positive inotropic effects.
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How is arteriolar vasoconstriction initiated in the heart?
Sympathetic release of noradrenaline activated Alpha1-adrenoreceptors to stimulate phosphlipase C and IP3 production via Gq. IP3 releases ER Ca and initiates a contractile response
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What stimulates Mu-opiod receptors?
endogenous opiods or by analgesics eg morphine to couple G-alpha1 proteins
257
How do G proteins reduce neurotransmitter release?
G- beta-gama subunits, liberated from teh heterotrimer interact with VOCCs to reduce Ca entry, reducing neurotransmitter release
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How do drugs exert their effects?
Bind to a target (Mostly proteins)
Mostly reversibly
Binding by association and dissociation rates
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Name the 2 most common targets for drugs
Enzymes (47%)
| GPCRs (30%)
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What are 3 other drug targets?
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Ion channels
Transporters
Nuclear hormone receptors
Receptors
Integrins
Misc
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When do drugs have the same concentration of molecules?
When they have the same molar concentrations. NOT WHEN THEY ARE OF EQUAL WEIGHT
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How many particles does 1 mole contain?
6 x10^23
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What does "1 molar solution" mean?
1 mole in 1 litre
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Define Affinity
Likelihood of a ligand binding to its target
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Define efficacy
Likelihood of activation
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What drugs have both affinity and efficacy?
agonists
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What drugs have affinity but no efficacy?
antagonists
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What is a radioligand and why might one be used?
A radioactive ligand often used to obtain information on binding
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What does Bmax show?
Maximum binding capacity. This gives information about the number of receptors
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What is Kd?
dissociation constant. A measure of affinity. The concentration needed for 50% occupancy
(lower Kd, higher affinity)
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What type of graph is used to measure response in cells/tissue?
concentration response curves
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When is a dose response curve used?
to measure response in a whole animal
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What is Emax?
Maximum response
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What is EC50?
Effective concentration giving 50% of the maximal response. A measure of potency.
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What is potency?
A combination of affinity and efficacy. The number of receptors also governs potency
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Do agonists with the same Emax have the same efficacy?
No, may have different affinity
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How can less than 100% receptor occupancy give a 100% response?
Spare receptors are present (receptor reserve) - more receptors than required to produce a maximal response.
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What influences the relationship between receptor occupancy and response?
transduction system/amplification produced by second messengers and the properties of the tissue
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What do spare receptors do?
increase sensitivity allowing for response at low conc of agonist. No. of receptors therefore has an effect on potency
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What drug is used as pain relief and recreationally?
Opioid (heroin)
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What negative side effects can opioid have?
Respiratory depression leading to death
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How do opioids work in the body?
Primarily through Mu-Opioid receptors (GPCR)
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What is a partial agonist of the same receptors of which morphine is a full agonist?
Buprenorphine. Higher affinity, lower efficacy
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Why is buprenorphine sometimes a better option than morphine?
Less respiratory depression. Used if adequate pain control as it is only a partial agonist
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What are the 3 different types of antagonists?
Reversible competitive
Irreversible competitive
Non-competitive
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What does reversible competitive antagonism rely on?
Dynamic equilibrium between ligand and receptors
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What is the most commonly used type of antagonist?
Reversible competitive
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How can the effect of competitive antagonists be overcome?
Increased amount of agonist - graph shifts left
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Give an example of a reversible antagonist
Naloxone. High affinity for Mu-opioid receptors. Reverses Mu-opioid receptor respiratory depresssion. High affinity- competes effectively
290
What is an irreversible competitive antagonist?
Agonists dissociates slowly or not at all
291
How does irreversible antagonist affect a graph of agonist against response?
Shifts the graph to the right as the antagonist is increased before decreasing the max response as the antagonist binds irreversibly and not enough receptors free to illicit a max response
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Give an example of an irreversible antagonist.
Phenoxybenzamine. Non selective alpha1-adrenoreceptor used in hypertension episodes in phenochromocytoma
293
How does a non-competitive antagonist work?
allosterically binds to a receptor
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How does irreversible antagonist affect a graph of agonist against response?
Shifts the graph to the right as the antagonist is increased before decreasing the max response as the antagonist binds irreversibly and not enough receptors free to illicit a max response
295
Give an example of an irreversible antagonist.
Phenoxybenzamine. Non selective alpha1-adrenoreceptor used in hypertension episodes in phenochromocytoma
296
How does a non-competitive antagonist work?
allosterically binds to a receptor
297
Define Pharmacokinetics
What the body does to the drug
298
What are the possible formulations of a drug?
Solid
| Liquid
299
What are the sites of administration of a drug?
Local (eye, skin, inhalation etc)
Systemic - Enteral (sublingual, oral, rectal), Parental (subcutaneous, intramuscular, IV injection, inhalation, transdermal)
300
Define oral bioavailability
The proportion of a dose given orally (or by any other route other than IV) that reaches the systemic circulation in an unchanged form.
301
How can oral bioavailability be expressed?
Amount - Measured by area under the curve of blood drug
| Rate - Measured by peak height and rate of rise of drug level in blood
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What is the therapeutic Ratio?
Max. Tolerated Dose/Min. Effective Dose
| Lethal Dose to 50%) LD50/ED50 (Effective Dose in 50% of people
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What is the first pass effect?
Substances absorbed from the lumen enter the venous blood, which drains into the hepatic portal vein and is transported directly to the liver which is the main site of drug metabolism as it contains all of the necessary enzyme systems -> drugs absorbed from the lumen extensively metabolised in this first pass through the liver
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What types of drug administration avoid the first pass effect?
parenteral
sublingual
rectal
90% of oral dose is usually metabolised in first pass
305
What is drug distribution?
The theoretical volume into which a drug has distributed assuming this is occurring instantly
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How do you calculate drug distribution?
Amount given/plasma conc at time 0
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In the plasma, what drugs exert their effect?
Free level of drugs not the total
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When is protein interaction of drugs important?
When it is highly bound to albumin
Has a small volume of distribution
Has a low therapeutic index
309
What are Class I Drugs?
Drugs used at a dose that is much lower than the number of albumin binding sites
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What are Class II Drugs?
Drugs used at a dose that is greater than the number of available binding sites
311
What happens when Class I and II are administered together?
Class I is displaced by Class II, raising the levels of the object drug and therefore, higher risk of toxicity
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What are first order kinetics?
Rate of elimination is proportional to drug level. Constant fraction of drug eliminated in unit time. Half life can be defined as the rate of decline of plasma drug level is proportional to drug level
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What are zero order kinetics?
Rate of elimination is constant.
Enzyme is saturated
Steady state is reached within 5 half lives. If an immediate effect is necessary, a loading dose is therefore needed
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On what graph is a first order kinetic linear?
Log Y axis plotted against time
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On what graph is a zero order kinetic linear?
Linear Y axis scale plotted against time
316
When do drugs tend to have zero order?
At high doses - metabolism is constant and independent of dose
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When do drugs tend to have first order?
Low doses - metabolism is proportional to dose
318
Compare the response of 1st and zero order kinetics
1st - predictable therapeutic response from dose increases
| zero - therapeutic response can suddenly escalate quickly as elimination mechanisms saturate
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What does Phase I do to a drug?
Exposes/adds a reactive group if the parent molecule is unreactive, generating an intermediate
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What reactions commonly occur in phase I of drug metabolism?
Oxidation
Reduction
Hydrolysis
321
What complex enzyme system is required for phase I?
```
cytochrome P450 (CYP) and a high energy cofactor, (NADPH)
Enzymes are inducible and inhibitable
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What happens in phase II of drug metabolism?
reactive intermediate is conjugated with a polar molecule to form a water soluble complex - conjugation
323
What are common conjugates for phase II drug metabolism?
Glucoronic acid is the most common conjugate as it is an available by-product of cell metabolism.
Sulphate ions and glutathione also used.
324
What specific enzymes and cofactors does phase II drug metabolism require?
uridine diphosphate glucuronic acid (UDPGA)
325
What effects drug excretion by the kidneys?
Only free, unbound drug is filtered through the glomerular tuft
Drugs can be actively secreted by the tubule
Urine pH can determine how much of the drug is excreted
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How does pH effect the excretion of drugs in the kidney?
Weak acidic drugs, making the urine more alkaline will make the drug ionised, so there will be less tubular absorption because the charged drugs stay in the tubule lumen. (Opposite for weak alkali)
327
What does the ANS do?
Controls all involuntary (vegetative) functions
328
What type of nerves does the ANS use?
Entirely efferent but regulated by afferent inputs
329
What does the sympathetic nervous system do?
Responds to stressful situations - fight or flight
330
What does the parasympathetic nervous system do?
Regulates basal activities
331
Are ANS nerves myelinated or unmyelinated?
Myelinated pre, unmyelinated post
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Where do parasympathetic nerves originate?
Lateral horn of the medulla and sacral regions of the spinal cord
333
Where are the ganglia of the parasympathetic nervous system located?
in the tissues innervated by the postsynaptic fibres
334
WHere do sympathetic nerves originate from?
The lateral horn of the lumbar and thoracic cord
335
Where are the ganglia of the sympathetic nervous system located?
paravertebral chain close to the spinal cord
336
What is the neurotransmitter used in preganglionic neurones in the ANS?
Ach
Cholinergic neurons
Activate post-ganglionic nicotinic Ach receptors - ligand gated ion channels
337
What neurotransmitter is released by post-ganglionic parasympathetic neurons and what type of receptors does it act on?
Ach
Acts on muscarinic Ach receptors in the target tissue
GPCRs
338
What neurotransmitter is commonly released fro post-ganglionic sympathetic neurones?
Noradrenaline - noradrenergic
acts on alpha-adrenoreceptors and beta receptors (1 and 2 and beta 3)
GPCRs
339
What structures are innervated by cholinergic sympathetic post-ganglionic neurones?
Sweat glands
| Hair follicles
340
What are NANC transmitters?
Non-Adrenergic, Non-Cholinergic transmitters
May be co-released with either NA or Ach e.g. ATP, Nitric Oxide, Serotonin, Neuropeptides
Some ANS transmitters behave like this
341
What are chromaffin cells and how are they innervated?
Present in adrenal gland - neurons differentiate to form chromaffin cells. They can be considered as postganglionic sympathetic neurons that do not project to a target tissue. On sympathetic stimulation, release adrenaline in to the blood stream.
Innervated by preganglionic sympathetic neurons
342
What are some ANS disorders?
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Catecholamine disorders
Central autonomic disorders
Orthostatic intolerence syndrome
Paroxysmal autonomic syncopes
Peripheral autonomic disorders
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343
What steps neurotransmission are commonly exploited by drug actions?
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Degradation of transmitter
Interaction with post-synaptic receptors
Inactivation of transmitter
Re-uptake of transmitter
Interaction with pre-synaptic receptors
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344
What is acetylchoine made of?
Acetyl CoA and choline, catalysed by choline acetyltransferase
345
What is acetylcholine degraded to?
Acetate and choline by acetylcholine esterase
346
What are ganglion blockers?
Drugs that have actions selectively at autonomic ganglia - nicotinic acetylcholine receptors at autonomic ganglia and neuromuscular junction differ in structure
347
How many types of muscarinic acetylcholine receptors are there?
5 but there are relatively few subtype-selective mAchR agonist or antagonists.
348
What do AChE inhibitors do?
Enhance the actions of endogenously released ACh
349
Why do cholinergic drugs often associate with unwanted side effects?
Relative lack of selectivity
350
What is pilocarpine and when is it used?
Muscarinic ACh receptor agonist used to treat glaucoma
351
What is bethanechol?
Muscarinic ACh receptor agonist used to sttimulate bladder emptying
352
Name 3 drugs used to treat an overactive bladder
tolterodine
darifenacin
oxybutynin
Muscarinic ACh receptor agonists
Severe dry mouth affects compliance
353
WHat is tiotropium?
M ACh receptor antagonist used to treat chronic obstructive pulmonary disease
354
What is ipratropium?
M ACh antagonist used to treat some forms of asthma
355
What are varicosities?
Sites on the highly branched axonal network of hte post ganglionic sympathetic neurons which are specialised for Ca dependent noradrenaline release
356
How is noradrenaline removed from the synaptic cleft?
By noradrenaline transporter proteins (high affinity) or if not, re-captured by a lower affinity non-neuronal mechanism- actions terminated by re-uptake
357
What metabolises NA?
monoamine oxidase or catechol-O-methyltransferase if not taken up into vesicles in the pre-synaptic terminal
358
How is neurotransmitter release modulated?
Presynaptic G protein coupled receptors can regulate neurotransmitter release by inhibiting Ca dependent exocytosis - BG subunits inhibit specific types of voltage operated Ca channels reducingCa influx and neurotransmitter release
359
What are indirectly-acting sympathomimetic agents?
Taken up into noradrenergic synaptic vesicles where they cause noradrenaline to leak from the vesicle. Displaced noradrenaline leaks into the synaptic cleft by a mechanism other than Ca mediated exocytosis
360
What are uptake 1 inhibitors?
Selective noradrenaline re-uptake inhibitors - mostly work on the CVS. Peripheral actins tend to be unwanted side effects
