physiology and pharmacology Flashcards
drug targets:
proteins: receptors, enzymes, transport proteins, ion channels
nucleic acids: DNA, RNA
receptor =
a protein that is involved in the normal process of cell communication
embedded in the plasma membrane or within the cell cytoplasm or nucleus
enzymes as drug targets -
inhibitors - can be competitive/non competitive
e.g. acetylsalicyclic acid (asprin) is a non competitive inhibitor of cyclooxygenase
false substrates - drugs converted by enzymes to generate abnormal product
modification of pro-drugs - requires chemical modification before becoming active
transporters as drug targets -
inhibitor - block transport
e.g.cocaine inhibits noradrenaline
false substrate - drug competes with normal substrate leading to accumulation of an abnormal compound
ion channels as drug targets -
ion channel blockers - drug binds in the channel pore to affect ion permeation
e.g. tetrodotoxin blocks Na channels
ion channel modulators - bind to the ion channel protein to affect gating
receptors as drug targets -
agonists - ligand that binds and activates a receptor to produce a response
antagonist - ligand that binds to a receptor and prevents its activation by an agonist, lacks efficacy
nuclear receptors -
inside the nucleus, act as transcriptional regulators
- steroid hormones
- thyroid hormones
- vitamin D
- fatty acids
ligand gated ion channels -
- regulated by the binding of a specific agonist
- mediate signalling in nervous system
comparison:
ligand gated ion channel -
location: membrane
effector: ion channel
coupling: direct
e. g: nicotinic acetylcholine receptor, GABAa receptor
structure: oligomeric assembly of subunits surrounding central pore
comparison:
G protein coupled receptor -
location: membrane
effector: channel or enzyme
coupling: G protein or arrestin
e. g: muscarinic acetylcholine receptor, adrenoreceptor
structure: oligomeric assembly of subunits comprising 7 transmembrane alpha helices with intracellular G protein coupling domain
comparison:
receptor kinases -
location: membrane
efffector: protein kinases
coupling: direct
e. g: insulin, growth factors, cytokine receptors
structure: single transmembrane helix linking extracellular receptor domain to intracellular kinase domain
comparison:
nuclear receptors -
location: intracellular
effector: gene transcription
coupling: via DNA
e. g: steroid receptors
structures: monomeric structure with receptor and DNA binding domains
hydrophobic ligands -
passively diffuse across the cell membrane use intracellular receptors function as activators or repressors of genes
ligand regulated transcription factors -
control cell proliferation, development, metabolism and reproduction
nuclear receptor superfamily:
steroids - oestrogen, progesterone
non steroidal lipophilic regulators - fatty acids, thyroid hormones, vitamin D3
orphans - no ligand or ligand unknown
signal transduction pathways:
second messengers…
- diffusible signals
- bind target proteins to change conformation and activity
- highly localised diffusion field
e.g. calcium ions + cAMP
cAMP as a second messenger in signal transduction pathways…
cyclic adenosine monophosphate
- binds regulatory subunits of protein kinase A
- subunits dissociate relieving inhibition of PKA
calcium ions as a second messenger in signal transduction pathways…
- enter cell via ion channels or are released from the endoplasmic reticulum via ion channels
- IP3 (inositol triphosphate) is released from plasma membrane and stimulates the opening of the ion channels
- calcium can interact directly with effector proteins to induce conformational change or can act through intermediate calmodulin protein to modify the activity of the effector
signal transduction pathways:
protein phosphorylation…
= the addition of phosphate to specific sites on proteins as a regulatory mechanism in signal transduction
- changes bulk and charge of the amino acid residue side chain
- addition of a phosphate causes structural alteration
= changes protein activity
signal transduction pathways regulate activity of:
transcription factors - increase gene expression
translation factors - increase protein expression
proteolytic enzymes - decrease protein expression
cytoskeletal proteins - regulate motility and shape
cell proliferation and death effectors
G protein coupled receptors respond to…
neurotransmitters - acetylcholine, GABA
hormones - glucagon, adrenaline
large glycoproteins - thyroid stimulating hormones
G protein coupled receptors couple to…
heterotrimeric G protein which controls the activity of an effector protein
G proteins comprise of…
a family of membrane resident proteins that recognise activated GPCRs and pass on the message to the effector systems that generate a cellular response
the G protein consists of 3 subunits (alpha, beta, gamma) which are anchored to the membrane through attached lipid residues
G protein coupled receptors respond to ligands by…
changing conformation
GTP binding causes dissociation of beta/gamma subunits and release of GDP = leading to an active state
G proteins are regulated by…
GEFs = guanine nucleotide exchange factors
- stimulates G protein to assure it acheives active conformation (stimultes loss of GDP and gain of GTP)
G protein activation cycle:
1) when a GPCR is activated by an agonist, a conformational change occurs
2) coupling of the alpha subunit to an agonist occupied receptor causes the bound GDP to exchange with intacellular GTP
3) the alpha-GTP complex dissociates from the receptor and interacts with a target protein, leaving a beta/gamma complex which also activates a target protein
4) the GTPase activity of the alpha subunit is increased when the target protein is bound leading to hydrolysis of GTP TO GDP = the alpha subunit reunites with beta/gamma
different types of G protein…
Gαs = activates adenylyl cyclase
Gαi = inhibits adenylyl cyclase
Gαq = activates phospholipase Cβ - (IP3/DAG signalling)
Gαs subunit -
stimulates adenylyl cylcase causing increased cAMP production
β2 adrenergic receptors use Gαs to regulate smooth muscle tone
activated by cholera toxin which blocks GTPase activity thus preventing inactivation
Gαi subunit -
inhibits adenylyl cylcase causing decreased cAMP production
blocked by pertussis toxin which prevents dissociation of αβγ complex
Gαq subunit -
activates phospholipase C, increasing production of second messenger IP3 (inositol triphosphate) and DAG (diacylglycerol)
muscarinic M2 regulation of GIRK channel in heart regulated by Gβγ:
GIRK channel = G protein-coupled inwardly-rectifying potassium channel
when ACh binds to M2R - activation of Gα and dissociation of Gβγ
when Gβγ binds to an open GIRK - increase in K+ permeability hyperpolarises the membrane - reduces heart muscle contraction - reduces heart rate
desensitisation - terminating GPCR signalling…
- shape change reveals GEF and phosphorylation sites
- G protein coupled receptor kinase phosphorylate
- turn off ability for G protein to couple to GPCR
- binding site is created for β Arrestin
- desensitisation of receptor for cell surface
receptor tyrosine kinase:
- type of enzyme coupled receptor
- mainly control cell growth, movement and differentiation
- involved in development and tissue repair
- main ligands are growth factors
- ligand binding causes conformational change resulting in clustering
- intracellular kinase domains can act on each other
- activated by ligand induced oligomerisation
- receptor clustering activates the kinases by transphosphorylation mechanism
RTK get info to where it is needed in the cell by…
protein recruitment
= using second messengers and signalling cascades
specificity in RTK -
phosphorylated tyrosines are binding sites for recruitment of signalling proteins
specific binding domains mediate recruitment of signalling proteins to receptor tyrosine kinase
phosphotyrosine binds phosophotyrosine binding and SH2 (src homology 2) domains, which direct proteins to specific pY containing targets
adaptor proteins…
some signalling proteins have their own SH2/PTB domain but some proteins need linker proteins to bind pY, these linker proteins are called adaptor proteins
regulation of G proteins by receptor tyrosine kinase:
kinetics of GTPase activity and GTP/GDP axchange determine signalling activity
active = GTP bound inactive = GDP bound
SoS protein is a GEF for the small G protein Ras,
Ras activates the protein kinase Raf
GEFs stimulate GDP loss and GTP gain = conformational change opens binding interface for G protein
Adrenoceptors:
Mediate actions of…
adrenaline - a hormone released by chromaffin cells in the adrenal medulla
Noradrenaline - a neurotransmitter released by noradrenergic neurones in the central and autonomic nervous system
Noradrenaline as a neurotransmitter in the CNS:
- modulating attention, perception, learning, memory, arousal
- brainstem noradrenergic nuclei like noradrenergic locus coeruleus sends axonal projections that innervate the cortex, amygdala, hippocampus, hypothalamus, spinal cord
Noradrenaline aw a neurotransmitter In the ANS:
Sympathetic activity increases at times of stress preparing the body for fight/flight
Sympathetic axons terminally branch to produce a network of noradrenergic varicosities each able to release noradrenaline at the post ganglionic neuroeffector junction
Key stems in adrenergic transmission:
1) synthesis
2) storage/compartmentation
3) release
4) signal transmission
5) re-uptake
6) metabolism
1) synthesis :
- noradrenaline is synthesised from the amino acid tyrosine within the nerve terminal
- the rate limiting enzyme for noradrenaline synthesises tyrosine hydroxylase
- tyrosine is converted to DOPA and then to dopamine in the cytoplasm. Dopamine is taken up into vesicles where it is converted to noradrenaline
- adrenal medulla chromaffin cells express phenylethanolamine N-methyltrransferase which converts noradrenaline to adrenaline
2) storage / compartmentation:
Noradrenergic terminal vesicles possess a dopamine/noradrenaline transporter that allows accumulation of noradrenaline at high concentration inside vesicles
3) Release:
Noradrenaline release is triggered by depolarisation of the nerve terminal, Ca2+ influx and vesicle fusion with the pre synaptic plasma membrane
4) signal transmission :
Released noradrenaline can bind adrenoceptors locates either pre or post synaptically
5) re uptake:
Noradrenaline is rapidly from the synaptic cleft by a high affinity uptake 1. Any noradrenaline evading this mechanism is cleared by uptake 2
6) metabolism:
Within the terminal cytoplasmic noradrenaline can be metabolised by monoamine oxidase (MAO), catechol-o-methyltransferase (COMT) and aldehyde dehydrogenase (reductase activities)
Alpha-methyltyrosine -
Blocks tyrosine hydroxylase, blocking De novo noradrenaline synthesis
Alpha-nethylnoradenaline differs from noradrenaline…
Is more resistant to metabolism by MAO (monoamine oxidase)/ COMT (catechol-o-methyltransferase) compared to noradrenaline = over time accumulates to become a major constitutent of vesicles
Alpha-methylnoradrenaline is a selective agonist at some receptors (false transmitter = outcompetes noradrenaline
alpha-mehylDOPA -
analogue of DOPA, which is converted to alpha-methyldopamine (by DOPA decarboxylase) and alpha-methylnoradrenaline (by dopamine beta-hydroxylase)
= causes accumulation of a false transmitter = outcompetes noradrenaline = decreased transmission at sympathetic neuro-effector junctions
what are the roles of presynaptic receptors:
Presynaptic receptors often respond to the transmitter released by the terminal (autoreceptors) - This can result in an increased or decreased likelihood of further transmitter release.
e.g. Presynaptic α2-adrenoceptors
= act as inhibitory autoreceptors decreasing noradrenaline release from the terminal.
= are Gi/o-coupled GPCRs that can decrease adenylyl cyclase activity and decrease N/P/Q-type voltage-gated Ca2+ channel opening.
α-adrenoceptors agonist potency ranking:
adrenaline ≥ noradrenaline > > isoprenaline
β-adrenoceptors agonist potency ranking:
isoprenaline > adrenaline ≥ noradrenaline
α1-adrenoceptor mediated response example…
arterial vasoconstriction
β-adrenoceptor-mediated response example…
atrial contraction
β2-adrenoceptors..
bronchodilatation, vaso-relaxation
β1, β2 and β3 adrenoceptors couple positivelt to…
adenylyl cyclase via Gs proteins
α2A, α2B and α2C adrenoceptors couple negatively to….
adenylyl cyclase via Gi/o proteins
α1A, α1B and α1D adrenoceptors couple positively to …
phospholipase C via Gq/11 proteins
Different adrenoceptor subtypes are expressed by different…
In the heart, β1-adrenoceptors mediate….
In the airways, β2-adrenoceptors mediate….
…cells/tissues allowing specific, characteristic responses to noradrenaline/adrenaline
…sympathetic/noradrenaline positive inotropic and positive chronotropic effects
…bronchodilatory effects (physiologically, more likely to be mediated by blood-borne adrenaline)
Positive inotropic effects in the heart:
Both blood-borne adrenaline and sympathetically released noradrenaline can interact with ventricular β1-adrenoceptors to increase the force of contraction (increases [Ca2+])
ADRENOCEPTOR AGONIST THERAPEUTICS:
Cardiovascular System -
β-adrenoceptor agonists (e.g. adrenaline, isoprenaline) and β1-selective adrenoceptor agonists (e.g. dobutamine)…
are used in the acute management of heart failure, cardiac arrest and heart ‘block’ (dysfunctional electrical conduction )
ADRENOCEPTOR AGONIST THERAPEUTICS:
Respiratory System -
Inhaled β2-selective adrenoceptor agonists (e.g. salbutamol, terbutaline) are used to bring about airways bronchodilation
ADRENOCEPTOR AGONIST THERAPEUTICS:
Anaphylactic Reactions -
Intramuscular injection of adrenaline (epipen®) is used as a first-line treatment in acute anaphylactic (type 1 hypersensitivity) reactions
ADRENOCEPTOR ANTAGONIST THERAPEUTICS:
Cardiovascular System/Hypertension -
α1-adrenoceptor antagonists (e.g. prazosin, doxazosin) are used to treat mild hypertension
β-adrenoceptor antagonists (e.g. propranolol, atenolol) are used to treat a variety of cardiovascuar disorders, including hypertension, angina pectoris and cardiac dysrhythmias
congestive heart failure
Use a β-blocker (e.g. bisoprolol or carvedilol), in addition to an ACE inhibitor and diuretic, as this has proven additional mortality and morbidity benefits
