Pharmacology and prescribing Flashcards
what is the FP10? who can issue them?
a prescription that can be issued by GPs, nurses and pharmacist prescribers, supplementary prescribers or hospital doctors in England
what is the colour of the GP FP10?
green
what is the colour of the FP10D?
yellow, issued by dentists
what is the colour of the FP10MDA?
blue, used for drugs such as methadone
what is the colour of the FP10P, PN, SP, or CN?
purple or green, used by prescribers such as nurses or pharmacists
what should be included on the FP10 prescription?
- prescriber’s signature
- prescriber’s address: practice address, usually already printed on the FP10. contain a number to identify the prescriber.
- date
- patient’s details
- information about the product supplied
what does the patient do with the FP10 and what payment is made?
- can be taken to any chemist/pharmacy in England
- pay a prescription charge for each which is currently £8.60 unless exempt
what are the protein targets for drug action on mammalian cells?
- receptors
- ion channels
- enzymes
- transporters (carrier molecules)
what is colchicine used for? what is the mechanism of its action?
- used to treat arthritic gout attacks
- interacts with the structural protein tubulin
what is an example of an immunosuppressive drug? how do most of them work?
- ciclosporin
- bind to cytosolic proteins known as immunophilins
how can therapeutic antibodies work?
act by sequestering cytokines
what are targets for chemotherapeutic drugs?
- aim is to suppress invading microorganisms or cancer cells
- targets include DNA and cell wall constituents, and other proteins
what are receptors?
sensing elements in the system of chemical communications that coordinates the function of all different body cells and chemical messengers
what are orphan receptors?
the mediator is still unknown
when are endogenous mediators usually discovered?
years before the receptor was characterised pharmacologically and biochemically
how can receptors be characterised? what are examples of receptors that were identified later?
- on basis of pharmacological and molecular characteristics
- cannabinoid and opioid receptors were discovered later
what are ion channels?
gateways in cell membranes that selectively allow the passage of particular ions, and are induced to open or close by many mechanisms
what are types of ion channels?
- ligand gated channels
- voltage gated channels
what are ligand gated channels?
- open only when one or more agonist molecules are bound
- properly classified as receptors, since agonist binding is needed to activate them
what are voltage gated channels?
ion channels which are gated by changes in the transmembrane potential rather than by agonist binding
in what ways can drugs affect ion channel function?
- by binding to the channel protein itself, either to the orthosteric or allosteric sites
- indirect interaction, involving G protein and other intermediaries
- by altering the level of expression of ion channels on the cell surface
what are orthosteric and allosteric sites?
- orthosteric: ligand-binding site of ligand-gated channels
- allosteric: other, non ligand-binding site
what is an example of drug molecules plugging the channel physically?
- action of local anaesthetics on the voltage-gated sodium channel
- drug molecule plugs the channel physically, blocking ion permeation
what are examples of drugs that bind to allosteric sites on the channel protein and thus affect channel gating?
- benzodiazepine tranquilisers: bind to a region of the GABA(a) receptor-chloride channel complex (ligand gated channel) that is distinct from the GABA binding site and facilitates the opening of the channel by GABA
- vasodilator drugs of the dihydropyridine type, which inhibit opening of L-type calcium channels
- sulfonylureas, used in treating diabetes, which act on ATP-gated potassium channels of pancreatic beta cells, enhancing insulin secretion
what is the effect/action of benzodiazapene transquilisers?
bind to a region of the GABA(a) receptor-chloride channel complex (ligand gated channel) that is distinct from the GABA binding site and facilitates the opening of the channel by GABA
what is the effect/action of dihydropyridine type drugs?
- vasodilation
- inhibit opening of L-type calcium channels
what is the effect/action of sulfonylureas?
used in treating diabetes; act on ATP-gated potassium channels of pancreatic beta cells, which enhances insulin secretion
what does indirect action between a drug and an ion channel involve?
G protein and other intermediaries
what is the action of gabapentin?
reduces the insertion of neuronal calcium channels into the plasma membrane
what effect do agonists/inverse agonists have on receptors?
- direct: ion channel opening/closing
- transduction mechanisms: enzyme activation/inhibition, ion channel modulation, DNA transcription
what effect do antagonists have on receptors?
- no effect
- endogenous mediators blocked
what effect do blockers have on ion channels?
permeation blocked
what effect do modulators have on ion channels?
increased or decreased opening probability
what effect do inhibitors have on enzymes?
normal reaction inhibited
what effect do false substrates have on enzymes?
abnormal metabolite produced
what effect do prodrugs have on enzymes?
active drug produced
what effect does an inhibitor have on transporters?
transport blocked
what effect does a false substrate have on transporters?
abnormal compound accumulated
what is an example of a drug molecule acting as a competitive inhibitor of the enzyme?
captopril, acting on ACE
what is an example of irreversible and non-competitive binding to an enzyme?
aspirin, acting on cyclo-oxygenase
what is a false substrate?
drug molecule undergoes chemical transformation to form an abnormal product that subverts the normal metabolic pathway
what is an example of a false substrate acting on an enzyme?
- fluorouracil, anticancer drug
- replaces uracil as an intermediate in purine biosynthesis but cannot be converted into thymidylate, thus blocking DNA synthesis and preventing cell division
what is a prodrug?
drugs may require enzymatic degradation to convert them from an inactive form to an active form
what is an example of prodrug conversion?
enalapril is converted by esterases to enalaprilat, which inhibits ACE
what does drug toxicity often result from?
- enzymatic conversion of the drug molecule to a reactive metabolite
what are ABC transporters?
- ATP-binding cassette transporters
- hydrolysis of ATP provides energy for transport of substances against their electrochemical gradient
what are MDR transporters?
multi-drug resistance transporters
- eject cytotoxic drugs from cancer and microbial cells, conferring resistance to these therapeutic agents
what are symports and antiports?
- symports: transport of organic molecules is coupled to the transport of ions in the same direction
- antiports: transport of organic molecules is coupled to the transport of ions in the opposite direction
what are the four receptor types?
- ligand-gated ion channels
- G protein coupled receptors (metabotropic receptors or 7 transmembrane receptors)
- kinase-linked and related receptors
- nuclear receptors
what is the basic function, time scale and examples of ligand-gated ion channels (ionotrophic receptors)?
function: influx of ions -> hyperpolarisation/depolarisation -> cellular effects
time scale: milliseconds
examples: nicotinic ACh receptor
what is the basic function, time scale and examples of G protein coupled receptors (metabotrophic)?
function: binding of ligand to receptor leads to increase/decrease in second messenger production or ion influx. Second messengers lead to Ca2+ release, protein phosphorylation or other. ion influx -> change in excitability -> cellular effects
time scale: seconds
example: muscarinic ACh receptor
what is the basic function, time scale and example of kinase-linked receptors?
function: binding of ligand to receptor/enzyme -> protein phosphorylation -> gene transcription -> protein synthesis -> cellular effects
time scale: hours
example: cytokine receptors
what is the basic function, time scale and example of nuclear receptors?
function: binding of ligand to receptor within the nucleus -> gene transcription -> protein synthesis -> cellular effects
time scale: hours
example: oestrogen receptor
what are characteristics of receptors within a given family (heterogeneity)?
- generally occur in several molecular varieties/subtypes
- similar architecture
- significant differences in their sequences and pharmacological properties
where is the nicotinic acetylcholine receptor located?
skeletal NMJ
what are cys loop receptors?
receptors that have a large intracellular domain between transmembrane domains 3 and 4 containing multiple cystein residues
what are examples of cys-loop receptors?
- GABA
- glycine receptors
- 5-HT receptors
- nicotinic ACh receptor
what are types of ligand-gated ion channels?
- nicotinic ACh receptors
- ionotropic glutamate receptors
- purinergic P2X receptors
what is the molecular structure of the nicotinic ACh receptor?
- pentameric assembly of different subunits (2 x alpha)
- alpha, beta, gamma and delta subunits, each of molecular weight 40-58 kDa
- subunits show marked sequence homology, each containing four membrane-spanning alpha-helices
- two ACh binding sites, each at the interface between one of two alpha subunits and its neighbour
- each subunit spans the membrane four time, so there are 20 membrane-spanning helices surrounding a central pore
how must nicotinic ACh receptors be activated?
both binding sites must bind ACh molecules
where do the two ACh binding sites on nicotinic ACh receptors lie?
- at interface between one of two alpha subunits and its neighbour
- on the extracellular N-terminal region of the two alpha subunits
what forms the lining of the ion channel in nicotinic ACh receptors? what forms the constriction?
- one of the transmembrane helices (M2) from each of the five subunits forms the ion channel
- five M2 helices that form the pore are sharply kinked inwards halfway through the membrane, forming a constriction
what conformational change occurs when ACh molecules bind to a nicotinic ACh receptor?
- conformation change occurs in the extracelllar part of the receptor
- twists the alpha subunits, causing the kinked M2 segments to swivel out of the way
- this opens the channel
what does the channel lining of the nicotinic ACh receptor contain?
series of anionic residues, making the channel selectively permeable to cations
- primarily Na+ and K+, sometimes Ca2+
what is the location, effector, coupling, examples and structure of ligand-gated ion channels?
- membrane
- ion channel
- direct
- nicotinic ACh receptor, GABA(a) receptor
- oligomeric assembly of subunits surrounding the central pore
what is the location, effector, coupling, examples and structure of G-protein coupled receptors?
- membrane
- channel/enzyme
- G protein or arrestin
- muscarinic ACh receptors, adrenoreceptors
what is the location, effector, coupling, examples and structure of receptor kinases?
- membrane
- protein kinases
- direct
- insulin, growth factors, cytokines
- single transmembrane helix linking extracellular receptor domain to intracellular kinase domain
what is the location, effector, coupling, examples and structure of nuclear receptors?
- intracellular
- gene transcription
- via DNA
- steroid receptors
- monomeric structure with receptor and DNA binding domain
what is the structure of ionotropic glutamate receptors?
tetrameric
- pore is built from loops rather than transmembrane helices
what is the structure of P2X receptors?
trimeric
- each subunit has only two transmembrane domains
what is positive cooperativity?
binding of one agonist molecule to one site increasing the affinity of binding at the other site
what are examples of GPCRs?
- muscarinic AChRs
- adrenoceptors
- dopamine receptors
- 5-HT receptors
- opioid receptors
- peptide receptors
- purine receptors
how many GPCRs are encoded for by GPCRs?
~ 400
what was the first GPCR to be characterised?
beta adrenoceptor, which was cloned in 1986
what is the structure of GPCRs?
- single polypeptide chain, usually of 350-400 residues
- can be up to 1100 residues
- 7 transmembrane alpha helices
- extracellular N-terminal domain of varying length, and an intracellular C-terminal domain
what are the families of GPCRs?
A: rhodopsin family
B: secretin/glucagon receptor
family
C: metabotropic glutamate receptor/calcium sensor family
what receptors are in family A of GPCRs? what are their structural features?
- largest group
- receptors for most amine neurotransmitters, many neuropeptides, purines, prostanoids, cannabinoids
- short extracellular (N-terminal) tail
- ligand binds to transmembrane helices (amines) or to extracellular loops (peptides)
what receptors are in family B of GPCRs? what are their structural features?
- receptors for peptide hormones, including secretin, glucagon, calcitonin
- intermediate extracellular tail incorporating ligand-binding domain
what receptors are in family C of GPCRs? what are their structural features?
- small group
- metabotropic glutamate receptors, GABA(b) receptors, Ca2+ sensing receptors
- long extracellular tail incorporating ligand-binding domain
what is a fourth distinct family of GPCRs?
includes many receptors for pheromones but no pharmalogical receptors
what is the site of the molecule that couples to the G protein?
third cytoplasmic loop
- experiments show that deletion/modification of this section results in receptors that will still bind ligands but cannot associate with G proteins/produce responses
what can result in receptor desensitisation?
phosphorylation of serine and threonine residues on the C-terminal tail and other intracellular domains by intracellular kinases
where is the ligand-binding domain of small molecules in class A receptors?
buried in the cleft between the alpha helical segments within the membrane
- similar to the slot occupied by retinal in the rhodopsin molecule
where do peptide ligands bind?
e. g. substance P
- bind more superficially to the extracellular loops
what are protease-activated receptors? how can they be activated?
- four types identified
- many proteases e.g. thrombin, can activate PARs by snipping off the end of the extracellular N-terminal tail of the receptor to expose 5 or 6 N-terminal residues that bind to receptor domains in the extracellular loops
what is PAR-2 activated by?
- activated by a protease released from mast cells
- expressed on sensory neurons
- may play a role in inflammatory pain
how many times can a PAR molecule be activated?
only once
- because cleavage cannot be reversed, so continuous resynthesis of receptor protein is necessary
how is PAR inactivated?
- further proteolytic cleavage that frees the tethered ligand
- by desensitisation involving phosphorylation
- receptor is internalised and degraded, then replaced
what are G proteins? what is their role?
- family of membrane-resident proteins
- function is to recognise activated GPCRs and passes on the message to effector systems that generate a cellular response
- interact with guanine nucleotides, GTP and GDP
what subunits do G proteins consist of?
three: alpha, beta and gamma
what happens to G proteins during binding?
- guanine nucleotides bind to the alpha subunit, which has enzymatic (GTPase) activity, catalysing the conversion of GTP to GDP
- beta and gamma subunits remain together as a complex
what is the structure of the gamma subunit in the G protein?
anchored to the membrane through a fatty acid chain
- coupled to the G protein through a reaction known as prenylation
are G proteins diffusible?
they are freely diffusable in the plane of the membrane, so a single pool of G protein in a cell can interact with several receptors and effectors
what is the state of the resting G protein?
alpha-beta-gamma trimer
- may or may not be precoupled to the receptor
- GDP occupies the site on the alpha subunit
what happens to the G protein when a GPCR is activated?
- when activated by an agonist molecule, there is a conformational change involving the cytoplasmic domain of the receptor
- high affinity interaction of the trimer and the receptor within 50 ms
- bound GDP dissociates and is replaced with GTP
- this causes dissociation of the trimer, releasing alpha GTP and beta-gamma subunits
what are the active forms of the G protein?
- alphaGTP
- beta-gamma
what are the actions of the active forms of the G protein?
diffuse in the membrane and can associate with various enzymes and ion channels, causing activation of the target
what does G protein activation lead to?
- amplification: a single complex can activate several G protein molecules, and each can remain associated with the effector enzyme for long enough to produce many molecules of product (often second messenger)
what is often the product of G protein activation?
second messenger
when is signalling of G proteins terminated? what happens after this?
when the hydrolysis of GTP to GDP occurs through the GTPase activity of the alpha subunit
- resulting alphaGDP dissociates from the effector, then reunites with beta-gamma
what are RGS? how can they affect GPCR?
- regulators of G protein signalling proteins
- possess a conserved sequence that binds specifically to alpha subunits to increase their GTPase activity, thus hastening the hydrolysis of GTP and inactivating the complex
- inhibitory effect on G protein signalling
how many RGS proteins are there?
~ 20 cellular proteins
what causes increased GTPase activity of the alpha subunit?
binding of the target protein by alpha subunit and/or RGS proteins
what are the Galpha protein subtypes?
Ga(s) Ga(i) Ga(o) Ga(q) Ga(12)
what are associated receptors and functions of Ga(s) subunits? what is it activated/blocked by?
associated receptors
- many amine and other receptors
- e.g. catecholamines, histamine, serotonin
functions
- stimulates adenylyl cyclase, increasing cAMP formation
activated by cholera toxin, which blocks GTPase activity, thus preventing inactivation
what are associated receptors and functions of Ga(i) subunits? what is it activated/blocked by?
associated receptors
- amine and other receptors (e.g. catecholamines, histamine, serotonin)
- opioid/cannabinoid receptors
functions
- inhibits adenylyl cyclase, decreasing cAMP formation
blocked by pertussis toxin, which prevents dissociation of the alpha-beta-gamma complex
what are associated receptors and functions of Ga(o) subunits? what is it activated/blocked by?
associated receptors
- amine and other receptors (e.g. catecholamines, histamine, serotonin)
- opioid/cannabinoid receptors
functions
- ?
- limited effects of alpha subunit (effects mainly due to beta-gamma subunits)
blocked by pertussis toxin
occurs mainly in nervous system
what are associated receptors and functions of Ga(q) subunits? what is it activated/blocked by?
associated receptors
- amide, peptide and prostanoid receptors
functions
- activates phospholipase C, increasing production of second messengers of inositol triphosphate and diacylglycerol
what are the associated receptors and functions of Gbeta-gamma subunits?
associated receptors
- all GPCRs
functions
- activate K+ channels
- inhibit voltage-gated calcium channels
- activate GPCR kinases
- activate mitogen-activated protein kinase cascade
- interact with some forms of adenylyl cyclase and phospholipase Cbeta
how many subtypes of the alpha subunit of G proteins are there?
more than 20
what subtypes stimulate and inhibit adenylyl cyclase?
Gs and Gi, respectively
what do cholera toxin and pertussis toxin do to G proteins?
- catalyse a conjugation reaction (ADP ribosylation) on the alpha subunit of G proteins
- cholera toxin acts only on Gs and causes persistent activation
- pertussis toxin specifically blocks Gi and Go by preventing dissociation of the G protein trimer
- symptoms of cholera, e.g. excessive secretion of fluid from the GI epithelium are due to uncontrolled activation of adenylyl cyclase
what are the main targets of G proteins, through which GPCRs control aspects of cell function?
- adenylyl cyclase: responsible for cAMP formation
- phospholipase C: responsible for inositol phosphate and diacylglycerol formation
- ion channels, particularly Ca2+ and K+
- Rho A/Rho kinase, a system that regulates the activity of many signalling pathways controlling cell growth and proliferaton, smooth muscle contraction etc
- mitogen activated protein kinase, a system that controls many cell functions, e.g. cell division
what is cAMP?
cyclic 3’,5’ adenosine monophosphate
- nucleotide synthesised synthesised within the cell from ATP by adenylyl cyclase
how is cAMP produced/inactivation?
- produced continuously and inactivated by hydrolysis to 5’-AMP by phosphodiesterases
what acts on GPCRs to increase/decrease the catalytic activity of adenylyl cyclase?
- drugs, hormones and neurotransmitters
- change concentration of cAMPs
how many molecular isoforms are there of adenylyl cyclase?
10 different molecular isoforms
what is the action of cAMP?
activation of protein kinases, esp. protein kinase A
what does cAMP do in cardiac myocytes?
increased activity of voltage-gated calcium channels
- phosphorylation of these channels increases the amount of Ca2+ entering the cell during the action potential, thus increasing the force of contraction of the heart
what does cAMP do in smooth muscle?
- phosphorylates (and inactivates) myosin light chain kinase, which is required for contraction
- accounts for smooth muscle relaxation produced by many drugs that increase cAMP production in smooth muscle
what are examples of Gi receptors?
- certain types of mAChR (e.g. M2 receptor of cardiac muscle)
- a2 adrenoceptors in smooth muscle
- opioid receptors
what is an example of a drug that activates adenylyl cyclase directly?
forskolin
how many subtypes of PDEs are there?
11
- some (e.g. PDE3 and PDE4) are cAMP selective
- others (e.g. PDE5) are cGMP selective
what are weak inhibitors of PDEs?
methylxanthines (e.g. theophylline and caffeine)
what drug is selective for PDE4?
- rolipram (asthma)
- PDE4 is expressed in inflammatory cells
what drug is selective for PDE5?
- milrinone, used for heart failure
- PDE5 is expressed in heart muscle
what drug is selective for PDE6?
- sildenafil (Viagra)
- enhances the vasodilator effects of NO and drugs that release NO, whose effects are mediated by cGMP
how does cAMP regulate energy metabolism?
- increased lipolysis
- reduced glycogen synthesis
- increased glycogen breakdown
how does cAMP increase lipolysis?
protein kinase phosphorylates/activates lipase
how does cAMP reduce glycogen synthesis?
protein kinase phosphorylates/inactivates glycogen synthase
how does cAMP increase glycogen breakdown?
protein kinase phosphorylates/activates phosphorylase kinase -> activated phosphorylase b -> conversion of glycogen to glucose-1-phosphate
what is PIP2? what is its structure?
- phosphatidylinositol (4,5) bisphosphate
- member of the membrane phospholipid class of phosphoinositides
- additional phosphate groups attached to the inositol ring
what is PIP2 a substrate for? what can it break up into?
- membrane bound enzyme, phospholipase Cbeta
- splits it into diacylglycerol and inositol (1,4,5) triphosphate
what is DAG and IP3?
diacylglycerol
inositol (1,4,5) triphosphate
what mediates the activation of phospholipase Cbeta (PLCb)?
Gq
how is PIP2 reformed?
- DAG is phosphorylated to form phosphatidic acid
- IP3 is dephosphorylated and then recoupled with phosphatdic acid to form PIP2
what is IP3? what is its role?
- water-soluble mediator that’s released into the cytosol and acts on a ligand-gated calcium channel present on the membrane of the endoplasmic reticulum
- controls the release of Ca2+ from intracellular stores
what can IP3 be converted into?
inositol (1,3,4,5) tetraphosphate
- role is unclear but it may play a role in controlling gene expression
what is the action and behaviour of DAG?
- activates protein kinase C, which catalyses the phosphorylation of intracellular proteins
- highly lipophilic, remains within the membrane
- binds to a specific site on the PKC molecule, causing it to migrate from the cytosol to the cell membrane (activation)
how many subtypes are there of PKC?
- at least 10
- distinct cellular distributions and phosphorylate different proteins
- several activated by DAG and raised intracellular Ca2+
what can PKCs be activated by?
- DAG and raised Ca2+ levels
- phorbol esters
- arachidonic acid (generated by action of phospholipase A2 on membrane phospholipids)
what are phorbol esters?
highly irritant, tumour promoting compounds produced by certain plants
what do the various PKC isoforms act on?
different functional proteins
- ion channels
- receptors
- enzymes
- transcription factors
- cytoskeletal proteins
how do G proteins target ion channels?
- control ion channel function directly by mechanisms not involving second messengers
- done by beta-gamma subunits of Gi and Go proteins
- controls K+ and Ca2+ channels
what are examples of G proteins acting directly on ion channels?
in cardiac muscle: mAChRs enhance K+ permeability which hyperpolarises the cells and inhibits electrical permeability
in neurons: many inhibitory drugs reduce excitability by opening G protein-activated inwardly rectifying K+ channels or by inhibiting voltage activated N and P/Q type Ca2+ channels
what is the Rho/Rho kinase system?
- activated by certain GPCRs which couple to G proteins of the G12/13 type
- free Galpha subunit interacts with a guanosine nucleotide exchange factor, which facilitates GDP-GTP exchange at another GTPase, Rho
what are the active/inactive forms of Rho?
Rho-GDP (resting form) is inactive
when GDP-GTP exchange occurs, Rho is activated, which activates Rho kinase
what is the action of Rho kinase?
- phosphorylates many substrate proteins
- controls cellular functions, e.g. smooth muscle contraction/proliferation, angiogenesis and synaptic remodelling
- enhances hypoxia-induced pulmonary artery vasoconstriction, which is importnat in pathogenesis of pulmonary hypertension
what is an example of a Rho kinase inhibitor?
fasudil
what is the MAP kinase system?
- involves several signal transduction pathways that are activated by cytokines and growth factors acting on kinase-linked receptors and by ligands activating GPCRs
- coupling of GPCRs to familes of MAP kinases can involve alpha and beta-gamma subunits and Src and arrestins (GPCR desensitisation)
- controls processes involved in gene expression, cell division, apoptosis and tissue regeneration
what is homologous desensitisation?
restricted to the receptors activated by the desensitising agonist
what is heterologous desensitisation?
affects other GPCRs
what processes are involved in GPCR desensitisation?
- receptor phosphorylation
- receptor internalisaton (endocytosis)
how can GPCRs be phosphorylated?
- residues (serine and threonine) mainly in the C terminal cytoplasmic tail can be phosphorylated by specific membrane-bound GPCR kinases and PKA/C
how do specific GPCR kinases act?
- on receptor activation, GRK2 and GRK3 are recruited to the plasma membrane by binding to free G protein beta-gamma subunits
- GRKs then phosphorylate the receptors in their activated state
how does phosphorylation lead to homologous desensitisation?
- phosphorylated receptor serves as a binding site for arrestins
- arrestins are intracellular proteins that block the interaction between the receptor and G protein, leading to selective homologous desensitisation
what is arrestin? what is its function?
- intracellular protein which blocks the interaction between the receptor and G proteins
- produces homologous desensitisation of GPCRs
- arrestin binding targets the receptor for endocytosis in clathrin-coated pits
what happens to an internalised GPCR?
- dephosphorylated and reinserted into the plasma membrane (resensitisation)
- trafficked to lysosomes for degradation (inactivation)
what can phosphorylation by PKA and PKC at residues different from those targeted by GRKs lead to?
- impaired coupling between the activated receptor and the G protein; agonist effect is reduced
- leads to homologous or heterologous desensitisation
- receptors phosphorylated by second messenger kinases are probably not internalised and are reactivated by dephosphorylation by phosphatases when the agonist is removed
what are the subtypes of the GABAb receptor?
2 subtypes of the GPCR, encoded by different genes
- functional receptor consists of a heterodimer of the two
what do most GPCRs exist as?
- some are functional as monomers
- most can exist as homomeric or heteromeric oligomers
what are the targets of receptors and G proteins?
receptors -> guanylyl cyclase and G proteins
G proteins -> adenylyl cyclase, phospholipase C and ion channels
what is the effect of guanylyl cyclase?
produces cGMP
what is the effect of adenylyl cyclase?
produces cAMP
what is the effect of phospholipase C?
produces IP3 and DAG
what is the effect of cGMP?
- activates protein kinase G
- ion channels
what is the action of PKG?
affects contractile proteins and ion channels
what is the effect of cAMP?
- activates protein kinase A
- ion channels
what is the action of PKA?
- activates enzymes, transport proteins etc
- ion channels
what is the action of IP3?
increased [Ca2+], leads to:
- activated enzymes, transport proteins etc
- activated contractile proteins
- activated ion channels
what is the action of DAG?
activates arachidonic acid and protein kinase C
what do arachidonic acids produce?
eicosanoids
- released as local hormones
- affect ion channels
what does PKC do?
activates enzymes, transport proteins etc, contractile proteins and ion channels
what does increased free intracellular Ca2+ lead to?
initiates many events, including contraction, secretion, enzyme activation and membrane hyperpolarization
how do G proteins affect ion channels?
- opening potassium channels, leading to membrane hyperpolarisation
- inhibiting calcium channels, thus reducing neurotransmitter release
what forms arachidonic acid and eicosanoids?
phospholipase A2 (controlled by G proteins)
how can constitutively active receptors act?
- GPCRs may be spontaneously active in the absence of any agonist
- seen in beta adrenoceptor, where mutations in the third intracellular loop/overexpression of the receptor, results in constitutive activation
- native GPCRs can show constitutive activity when expressed in vitro
- histamine H3 receptor shows constitutive activity in vivo
what is the process of receptor coupling to MAP kinases?
receptor activation -> RAF1 -> MEK1 -> ERK1/2 -> altered gene expression
receptor activation -> ASK1 -> MKK4/7 + MKK3/6 -> JNK1-3 + p38 (respectively) -> altered gene expression
what is the process of arrestin coupling to MAP kinases? how can ERK be activated?
GRK acts on two Ps on receptor -> binding of ARR
ERK is activated by:
- binding of Src at the plasma membrane then binding of ERK to Src
- direct activation after internalisation of the receptor/arrestin complex; JNK3 can also be activated like this
what is the process of desensitisation and trafficking of GPCRs?
- on prolonged agonist activation of the GPCR, selective GRKs are recruited to the plasma membrane and phosphorylate the receptor
- arrestin binds and trafficks the GPCR to clathrin-coated pits for internalisation into endosomes in a dynamin-dependent process
- GPCR is dephosphorylated by a phosphatase (PP2A)
- either recycled and reinserted to the plasma membrane or trafficked to lysosomes for degradation
what is biased agonism?
ligand-dependent selectivity for certain signal transduction pathways relative to a reference ligand at the same receptor
what are RAMPs? how were they discovered?
receptor activity-modifying-proteins
- family of membrane proteins that associate with some GPCRs and alter their functional characteristics
- discovered in 1998 when the functionally active receptor for the neuropeptide calcitonin gene-related peptide consisted of a complex of a GPCR that by itself lacked activity, with another membrane protein RAMP1
- when CRLR was coupled with RAMP2, it was activated by adrenomedulin
what are modern characteristics of GPCRs?
- one gene, through alternative splicing, RNA editing etc, can give rise to more than one receptor protein
- one GPCR protein can associate with others, or with proteins e.g. RAMPs, to produce more than one type of functional receptor
- different agonists may affect the receptor in different ways and elicit qualitatively different responses
- the signal transduction pathway does not invariably require G proteins, and shows cross-talk with tyrosine kinase-linked receptors
what can kinase-linked receptors be activated by?
- protein mediators: growth factors and cytokines
- hormones: insulin and leptin (at level of gene transcription)
what is the general structure of kinase-linked receptors?
- large proteins
- single chain of up to 1000 residues with single membrane-spanning helical region
- links a large extracellular ligand-binding domain to an intracellular domain of variable size and function
- over 100 have been cloned
what are roles of kinase-linked receptors?
controlling cell division, growth, differentiation, inflammation, tissue repair, apoptosis and immune responses
what are the main types of kinase-linked receptors?
- receptor tyrosine kinases
- receptor serine/threonine kinases
- cytokine receptors
what are examples of receptor tyrosine kinase receptors?
- receptors for many growth factors, e.g. epidermal growth factor and nerve growth factor
- TLRs
- insulin receptor (more complex, dimeric structure)
what are examples of receptor serine/threonine kinases? what do they do?
- smaller class than RTKs
- phosphorylate serine and/or threonine residues rather than tyrosine
- example: transforming growth factor
what are examples of cytokine receptors? what do they do?
- lack intrinsic enzyme activity
- when occupied, they activate various tyrosine kinases, such as Jak (the Janus kinase)
- ligands include cytokines such as interferons and CSFs
how is phosphorylation and dephosphorylation of proteins accomplished?
by kinases and phosphatases, respectively
- enzymes which are subject to regulation dependent on their phosphorylation status
what happens when ligands bind to kinase linked receptors?
in many cases, dimerisation occurs
- association of two intracellular kinase domains allows a mutual autophosphorylation of intracellular tyrosine residues to occur
what is the function of phosphorylated tyrosine residues?
serve as high-affinity docking sites for other intracellular proteins that form the next stage in the signal transduction cascade
what is the growth factor pathway of kinase-linked receptors?
Ras/Raf/mitogen-activated protein (MAP) kinase pathway
what are the steps in the growth factor pathway of kinase-linked receptors?
- growth factor binds to receptor domain which is lined to an intracellular tyrosine kinase domain through a transmembrane alpha helix
- conformation change and dimerisation occurs
- tyrosine autophosphorylation occurs. SH2-domain protein (Grb2) binds to intracellular tyrosine kinase domain
- phosphorylation of Grb2
- activation of Ras GDP/GTP exchange
- activation of Raf, which phosphorylates Mek
- Mek phosphoryaltes MAP kinase
- MAP kinase phosphorylates various transcription factors, which leads to gene transcription
what is the cytokine pathway of kinase-linked receptors?
Jak/Stat pathway
what are the steps in the cytokine pathway of kinase-linked receptors?
- cytokine binds to receptor, which is linked via a transmembrane alpha helix to an intracellular Jak domain
- dimerisation and conformational change; activation of Jak
- phosphorylation of receptor and Jak
- binding and phosphorylation of SH2-domain protein
- dimerisation of Stat
- gene transcription
what are SH2 domain proteins? what do they do?
Src homology proteins; first identified in the Src oncogene product
- highly conserved sequence of about 100 amino acids
- form a recognition site for the phosphotyrosine residues of the receptor
- individual SH2 domain proteins bind selectively to particular receptors, so the pattern of events triggered is specific
what happens when the SH2 domain protein binds to the phosphorylated receptor?
- varies according to the receptor involved
- many SH2 domain proteins are enzymes
- some growth factors activate a specific subtype of phospholipase C (PLCgamma), causing phospholipid breakdown, IP3 formation and Ca2+ release
- may couple phosphotyrosine-containing-proteins with other functional proteins
- end result is to activate or inhibit, by phosphorylation, transcription factors that migrate to the nucleus and suppress/induce expression of genes
what are Stats?
- family of transcription factors
- SH2-domain proteins that bind to phosphotyrosine groups on the receptor-Jak complex
- are phosphorylated themselves
- when activated, Stat migrates to the nucleus and activates gene expression
what is PI3 kinase? what are they activated by? what does it do?
phosphatidylinositol-3-kinase
- enzyme family activated by GPCRs and RTKs
- attach a phosphate group to position 3 of PIP2 to form PIP3
- protein kinases, esp. PKB, have recognition sites for PIP3 and are thus activated, controlling apoptosis, differentiation, proliferation and trafficking, and NO synthase activation in the endothelium
what does guanylyl cyclase respond to?
binding of natriuretic peptides
- activated by dimerisation
- similar to RTKs
what is CaM kinase?
Ca2+/calmodulin-dependent kinase
what target proteins do kinase cascades affect?
- enzymes
- receptors
- ion channels
- transporters
- transcription factors
- contractile proteins
- secretory mechanisms
what are responses to phosphorylation of target proteins by kinases?
- physiological responses
- immune response
- apoptosis
- malignant transformation
- growth
- differentiation
what is the NR family?
nuclear receptor family
- receptors for hormones/substances present in the cytoplasm of cells and translocate into the nucleus after binding with ligand
- includes orphan receptors (receptors with no known well-defined ligands)
what are examples of nuclear receptors?
- receptors for steroid hormones e.g. oestrogen and clucocorticoids
- thyroid hormone T3
- fat soluble vitamins D and A (retinoic acid)
what is an example of an orphan receptor?
- retinoid X receptor (first to be described in the 1990s)
- cloned on the basis of its similarity with the vitamin A receptor
what does the vitamin A receptor bind?
vitamin A derivative 9-cis-retinoic acid
what are adopted orphan receptors?
those which have found binding partners
where can nuclear receptors be located?
- in cytosol and then translocate to nucleus
- in nuclear compartment
how many members are there of the nuclear receptor family?
48
what is the structure of nuclear receptors?
- heterogenous N terminal domain harbours the AF1 (activation function 1) site
- core DNA binding domain with ‘zinc fingers’
- hinge region
- ligand-binding domain, AF2 co-activator domain and HSP binding
- C-terminal extension
what does the heterogenous N terminal domain of the nuclear receptor do?
- displays most heterogeneity
- harbours the AF1 site
- AF1 site binds to other cell-specific transcription factors in a ligand independent way and modifies the binding or regulatory capacity of the receptor itself
- alternative splicing of genes may yield several receptor isoforms each with different N-terminal regions
what does the core domain of nuclear receptors do?
- highly conserved
- consists of structure responsible for DNA recognition and binding
- comprises two zinc fingers: cysteine rich loops in the amino acid chain held in place by zinc ions
- recognise and bind to hormone response elements in genes
- regulates receptor dimerisation
what are zinc fingers?
cysteine rich loops in the amino acid chain held in place by zinc ions
- in the core DNA binding domain of nuclear receptors
what are HREs?
hormone response elements
- located in genes that are regulated by nuclear receptors
- short (4/5 base pairs) sequences of DNA to which NRs bind to modify gene transcription
what does the hinge region in nuclear receptors do?
- highly flexible
- allows it to dimerise with other NRs
- dimerisation can produce molecular complexes with diverse configurations, able to interact differently with DNA
what does the C-terminal do?
- contains the ligand-binding module
- specific to each class of receptor
what does the AF2 region do?
- important in ligand-dependent activation
- generally highly conserved although absent in Rev-erbAalpha and Rev-erbAbeta, which regulate metabolism as mart of a circadian rhythm
where are HREs usually present?
usually symmetrically in pairs or half-sites
- may be arranged together in different ways (e.g. simple or inverted repeats)
how do HREs affect gene transcription?
- ligand-bound receptor recruits large complexes of other proteins including co-activators/repressors to modify gene expression through its AF1 and AF2 domains
what are examples of co-activators that are recruited by ligand bound nuclear receptors?
- enzymes involved in chromatin remodelling, e.g. histone acetylase/decetylase: regulate unravelling of DNA to allow access by polymerase enzymes
what are examples of co-repressors that are recruited by ligand bound nuclear receptors?
histone decetylase and other factors that cause the chromatin to become tightly packed, preventing further transcriptional activation
what are the classifications of nuclear receptors?
two main classes (I and II) and two other minor groups (III, IV)
what is class I of nuclear receptors?
endocrine steroid receptors
- glucocorticoid and mineralocorticoid receptors (GR and MR)
- oestrogen, progesterone and androgen receptors (ER, PR and AR)
what is the mechanism of action of class I nuclear receptors?
- after diffusion/transportation into the cell from blood, ligands bind their NR partner with high affinity
- form homodimers and translocate to the nucleus
- transactivate or transrepress genes by binding to positive or negative HREs
- once bound, the NR recruits other proteins to form complexes that promote transcription of multiple genes
where are class I NRs located?
in absence of their ligand, they’re predominantly located in the cytoplasm, complexed with head shock and other proteins and possibly reversibly attached to the cytoskeleton/other structures
what are the ligands for AR, ERalpha/beta, GRalpha, PR and MR nuclear receptors?
AR: testosterone ERalpha/beta: 17beta oestradiol GRalpha: cortisol, corticosterone PR: progesterone MR: aldosterone
what drugs are there for class I NRs?
- all natural and synthetic glucocorticoids, mineralocorticoids and sex steroids
- together with their antagonists (e.g. raloxifine, 4-hydroxy-tamoxifen and mifepristone)
what are class II NRs? what does this group include and what are their functions?
ligands are generally lipids already present to some extent within the cell
- peroxisome proliferator-activated receptor (PPAR): recognises fatty acids
- liver oxysterol receptor (LXR): recognises and acts as a cholesterol sensor
- farnesoid receptor (FXR)
- xenobiotic receptor (SXR): recognises foreign substances/drugs
- constitutive androstane receptor (CAR): recognises steroid androstane and some drugs
what is responsible for metabolising most prescription drugs?
CYP3A metabolises 60%
what is the action of class II NRs when they bind ligands?
- induce drug metabolising enzymes when they sense foreign material
- bind some prostaglandins, non steroidal drugs and the antidiabeteic thiazolidinediones and fibrates
what is the ligand binding/operation of class II NRs?
almost always operate as heterodimers with the retinoid X receptor (RXR)
what types of heterodimers can be formed by class II NRs?
- non-permissive heterodimer: can be activated only by RXR ligand itself
- permissive heterodimer: can be activated by retinoic acid itself or by its partner’s ligand
how do class II NRs affect gene transcription?
- generally bound to co-repressor proteins
- dissociate when the ligand binds and allows recruitment of coactivator proteins
- leads to changes in gene transcription
- tend to mediate positive feedback effects
what do class III NRs do?
- form homodimers
- can bind to HREs, which don’t have an inverted repeat sequence
what do class IV NRs do?
- function as monomers or dimers
- only bind to one HRE half site
what are ion channels?
consist of protein molecules designed to form water-filled pores that span the membrane, and can switch between open and closed states
what is the rate and direction of ion movement through the pore governed by? what is this a function of?
electrochemical gradient for the ion, which is a function of its concentration on either side of the membrane, and of the membrane potential
what are ion channels characterised by?
- selectivity for particular ion species, determined by the size of the pore and nature of its lining
- gating properties (the nature of the stimulus that controls the transition between open and closed states of the channel)
- molecular architecture
what are the main cation-selective channels selective for?
- Na+, Ca2+ or K+
- or non-selective and permeable to all three
what are anion channels mainly permeable to?
Cl-
what is an example of a ligand-gated ion channel that doesn’t respond to neurotransmitters?
responds to changes in the local environment instead
- TRPV1 channel on sensory nerves mediates pain-producing effect of chilli pepper ingredient capsaicin
- responds to extracellular protons when tissue pH falls in inflamed tissue, and to heat
what ligand-gated channels in the plasma membrane respond to intracellular signals rather than extracellular ones?
- calcium-activated potassium channels: open and hyperpolarise the cell when Ca2+ increases
- calcium-activated chloride channels
- ATP-sensitive potassium channels: open when intracellular ATP concentration falls because the cell is short of nutrients
- arachidonic-acid sensitive potassium channels
- DAG sensitive calcium channels
what are the functions of calcium-activated chloride channels?
expressed in excitable and non-excitable cells
- epithelial secretion of electrolytes and water
- sensory transduction
- regulation of neuronal and cardiac excitability
- regulation of vascular tone
where are ATP-sensitive potassium channels located?
- many nerve and muscle cells
- insulin-secreting cells
what are calcium release channels? what do they do?
- IP3 and ryanodine receptors
- class of ligand-gated calcium channels that are present on the endoplasmic or sarcoplasmic reticulum rather than the plasma membrane
- control the relase of Ca2+ from intracellular stores
where can calcium also be released from, apart from the endoplasmic and sarcoplasmic reticulum? what controls this?
- from lysosomal stores
- by nicotinic acid adenine dinucleotide phosphate activating two pore domain calcium channels
what are store operated calcium channels? what is their function?
- located in the plasma membrane
- open when intracellular Ca2+ stores are depleted; through interaction of a Ca2+ sensor protein in the ER membrane with a dedicated Ca2+ channel in the plasma membrane
- opening of channels allows cytosolic free Ca2+ conc to remain elevated even when intracellular stores are low
what are examples of cation channels?
6T1P
2T1P
4T2P
what are examples of 6T1P channels?
voltage gated K+ channels, TRP channels
what are examples of 2T1P channels?
inward-rectifying K+ channels, acid-sensing ion channels, epithelial Na+ channel, degenerins
what are examples of 4T2P channels?
resting K+ channels
what factors modulate the gating and permeation of both voltage-gated and ligand-gated ion channels?
- ligands that bind directly to various sites on the channel protein
- mediators and drugs that act indirectly, mainly by activation of GPCRs
- intracellular signals, particularly Ca2+ and ATP/GTP
what are drug-binding domains of voltage-gated sodium channels?
altered gating: GPCR ligands -> phosphorylation of channel
channel block (extracellular side): tetrodotoxin, saxitoxin, conotoxins
block of inactivation: veratridine, batrachotoxin, scorpion toxins, DDT, pyrethroids
channel block: local anaesthetics, antiepileptic drugs, antidysrythmic drugs
how are receptors regulated in the short and long term?
short term regulation: desensitisation
long term regulation: increase or decrease of receptor expression
what are the principal mechanisms in receptor malfunction?
- autoantibodies directed against receptor proteins
- mutations in genes encoding receptors, ion channels and proteins involved in signal transduction
what is the pathway of adrenaline and noradrenaline synthesis?
tyrosine -> dopa -> dopamine -> noradrenaline -> adrenaline
- final step only occurs in the adrenal medulla
what was the timeline of ANS discovery?
1860s: muscarine was shown to mimic the actions of nervous activation and atropine to oppose these actions
1905: Langley showed nicotine activated the NMJ and curare opposed this activation
1920s: vagal nerve stimulation slowed the heart and released a transferrable chemical that could slow (frog) hearts
what is the role of the ANS?
- peripheral ANS divides into sympathetic and parasympathetic branches (opposing effects)
- ANS conveys all outputs from the CNS to the body, except for skeletal muscular control
- regulatory roles in vascular, airway and visceral smooth muscle, exocrine secretions, control of heart rate, energy metabolism in the liver, links to immune system
what is the structure of the ANS/SNS?
somatic nervous system: neurone comes from the CNS to innervate muscle
ANS: two nerves in series, the pre- and post-ganglionic fibres
- parasympathetic ganglia are near their targets with short post-ganglionic nerves
- sympathetic ganglia are near the spinal cord with longer post-ganglionic fibres
what are effects of the parasympathetic division of the ANS?
- constricts pupil
- stimulates tear glands
- strong stimulation of salivary flow
- slows heart rate
- constricts bronchi
- stimulates digestive juice secretion
- stimulates intestinal motility
- contracts bladder
- stimulates erection
what are the effects of the sympathetic division of the ANS?
- dilates pupil
- tear glands maintain eye moisture
- inhibition of excess salivary secretion
- accelerates heart rate and constricts arterioles
- dilates bronchi
- inhibits stomach motility and secretion
- inhibits pancreas and adrenals
- inhibits intestinal motility
- relaxes bladder
- stimulates ejaculation
what neurotransmitters are released in sympathetic neurones? what receptors do they act on?
preganglionic neuron: cholinergic, ACh released at autonomic ganglion, acts on nicotinic receptor of postganglionic neuron
postganglionic neuron: adrenergic, norepinephrine (NE) released at target tissue which has adrenergic alpha or beta receptors
what neurotransmitters are released in parasympathetic neurones? what receptors do they act on?
preganglionic neuron: cholinergic, ACh released at autonomic ganglion, acts on nicotinic receptor of postganglionic neuron
postganglionic neuron: cholinergic, ACh released at target tissue which has cholinergic receptors (muscarinic)
what cranial nerves carry parasympathetic signals?
III (oculomotor), VII (facial), IX (glossopharyngeal) and X (vagus)
what are examples of dual parasympathetic and sympathetic innervation with opposing roles?
gut, bladder, heart
what is innervated only by sympathetic nerves?
sweat glands, blood vessels
what is innervated only by parasympathetic nerves?
bronchial smooth muscle
what do post-ganglionic sympathetic fibres release when innervating sweat glands?
ACh to stimulate muscarine receptors
what are NANCs? what are they used by?
non-adrenergic, non-cholinergic autonomic receptors
- used by enteric nervous system, parasympathetic and sympathetic system
what are examples of NANC use in parasympathetic and sympathetic systems?
parasympathetic: NO and vasoactive intestinal peptide
sympathetic: ATP and neuropeptide Y
what does nicotine stimulate?
all autonomic ganglia via specific ganglionic nicotinic receptors, activating both parasympathetic and sympathetic nervous systems
what does muscarine stimulate?
- from poisonous mushroom
- activates muscarinic receptors of the parasympathetic nervous system
- amenable to drug targeting
what are muscarinic receptors?
M1-5, GPCRs
where is M1 located?
mainly in the brain
where is M2 located?
- mainly in the heart
- activation slows the heart, so we can block these
what are examples of M2 blockers?
atropine for life-threatening bradycardias and cardiac arrest
where is M3 located? what does it cause?
- glandular and smooth muscle
- causes bronchoconstriction, sweating, salivary gland secretion
where is M4/5 located?
mainly in the CNS
what is an example of a muscarinic agonist? what could it be used to treat?
pilocarpine
- stimulates salvation: useful after radiotherapy, or in Sjorgren’s syndrome (PNS)
- contracts iris smooth muscle: used to treat glaucoma by facilitating drainage of aqueous humour (PNS)
what is a side effect of Pilocarpine/muscarinic agonists?
slow the heart
what can hyoscine be used for?
in palliative are, to antagonise parasympathetic driven secretions
where is atropine from?
deadly nightshade
how can muscarinic antagonists be used to treat bradycardia?
- prevent bradycardia and BP drop, dry secretions perioperatively
- treat bradycardias induced by excessive doses of beat blockers
- treat bradycardia in cardiac arrest
how can muscarinic antagonists be used to treat bronchoconstriction? what are examples of short/long acting ones?
- in the airway, drugs block the M3 receptor (called anti-cholinergics or anti-muscarinics)
- short acting: ipratropium bromide (atrovent)
- long acting: LAMAs e.g. tiotropium, glycopyrrhonium
how is selectivity of muscarinic antagonists in treatment of bronchoconstriction controlled?
- selectivity by drug delivery mechanisms (inhalers)
- receptor selectivity (e.g. tiotropium relatively selective for M3 receptors)
what other muscle roles do anticholinergics have?
- treating overactive bladders (e.g. solifenacin)
- short acting ones open up the pupil to allow eye examination
- act on parasympathetic fibres in intestinal colic and spasm: IBS (mebeverine) and palliative care (hyoscine)
what roles do antimuscarinics and anticholinergics have outside the autonomic system?
- ACh signalling involved in memory: anticholinergics worsen memory, and acetylcholinesterase inhibitor may treat dementia
- anti-emetic actions (e.g. hyoscine for travel sickness)
how can anticholinergics affect memory?
worsen memory
- acetylcholinesterase inhibitors may treat dementia
what is muscarine the active principle of?
the posionous mushroom Amanita muscaria
what do larger doses of ACh produce after muscarinic effects have been blocked by atropine?
nicotinic-like effects, including:
- stimulation of all autonomic ganglia
- stimulation of voluntary muscle
- secretion of adrenaline from the adrenal medulla
- initial rise in BP due to stimulation of sympathetic ganglia and consequent vasoconstriction
- secondary rise in BP due to secretion of adrenaline
what do small and medium doses of ACh produce?
transient fall in BP due to arteriolar vasodilation and slowing of the heart: muscarinic effects that are abolished by atropine
what effect does ACh have on vascular endothelial cells?
release NO, which relaxes smooth muscle to cause generalised vasodilation
what do the muscarinic actions of ACh correspond to? are there any exceptions?
to those of ACh releaesd at postganglionic parasympathetic nerve endings
exceptions:
- ACh causing generalised vasodilation even though most blood vessels have no parasympathetic innervation
- ACh evokes secretion from sweat glands, which are innervated by cholinergic fibres of the sympathetic nervous system
what do the nicotinic actions of ACh correspond to?
to those of ACh acting on autonomic ganglia of the sympathetic and parasympathetic systems, the motor endplate of voluntary muscle and the secretory cells of the adrenal medulla
what are the main classes of nicotinic ACh receptors?
muscle, ganglionic and CNS types
what is the structure of nAChRs?
- pentameric
- five subunits are similar in structure
what are the different routes of administration for drugs?
oral (po) intravenous (iv) rectal (pr) subcutaneous (sc) intramuscular (im) intranasal (in) topical (top) sublingual (sl) inhaled (inh) nebulised (neb)
how are the 8 week immunisations administered?
mainly intramuscular (im) rotavirus is administered orally (po)
what are the vaccines that are given at 8 weeks?
- 5 in 1 vaccine (diphtheria, tetanus, whooping cough (pertussis), polio and haemophilus influenza type b)
- pneumococcal vaccine
- rotavirus vaccine
- Men B vaccine
how can paracetamol be administered?
orally (po), per-rectum (pr), intravenous (iv)
what are the members of the nAChRs subunit family?
- five subunits that form the receptor-channel complex are similar in structure
- so far 17 different members have been identified and cloned, designated alpha (10 types), beta (4 types), gamma, delta and e (one of each)
what is the structure of nAChR subunits?
each possess 4 membrane spanning helical domains
- one of these helices (M2) from each subunit defines the central pore
what do nAChR subtypes generally contain?
both alpha and beta subunits, the exception being the homomeric (alpha7)5 subtype found in the bran
where do the two binding sites for ACh on nAChRs reside?
- interface between the extracelluar domain of each of the alpha subunits and its neighbour
what is the muscle type nAChR?
(α1)2β1δε
what is the ganglion type nAChR?
(α3)2(β4)3
what is the CNS type nAChR?
(α4)2(β2)3
(α7)5
what are the main synaptic locations of nAChR subtypes?
(α1)2β1δε: skeletal NMJ, mainly postsynaptic
(α3)2(β4)3: autonomic ganglia, mainly postsynaptic
(α4)2(β2)3: many brain regions; pre- and postsynaptic
(α7)5: many brain regions; pre- and postsynaptic
what are the membrane responses of nAChR subtypes?
(α1)2β1δε: excitatory; increased cation permeability, mainly Na+ and K+
(α3)2(β4)3: excitatory; increased cation permeability, mainly Na+ and K+
(α4)2(β2)3: pre and postsynaptic excitation; increased cation permeability, mainly Na+ and K+
(α7)5: pre and postsynaptic excitation; increased cation permeability; produces large Ca2+ entry, evoking transmitter release
what are the agonists of nAChR subtypes?
(α1)2β1δε: acetylcholine, carbachol, succinylcholine
(α3)2(β4)3: acetylcholine, carbachol, nicotine, epibatidine, dimethylphenylpiperazinium
(α4)2(β2)3: nicotine, epibatidine, acetylcholine, cytosine, varenicline
(α7)5: epibatidine, dimethylphenylpiperazinium, varenicline
what are the antagonists of nAChR subtypes?
(α1)2β1δε: tubocurarine, pancuronium, atracurium, vecuronium, alpha-bungarotoxin, alpha-conotoxin
(α3)2(β4)3: mecamylamine, trimetaphan, hexamethonium, alpha-conotoxin
(α4)2(β2)3: mecamylamine, methylaconitine
(α7)5: alpha-bungarotoxin, alpha-conotoxin, methylaconitine
what do muscarinic receptors couple with? what do they activate?
- odd numbered muscarinic receptors (M1, M3 and M5) couple with Gq to activate the IP3 pathway
- even numbered receptors (M2 and M4) open potassium channels causing membrane hyperpolarisation and acting through Gi to inhibit adenylyl cylcase and reduce intracellular cAMP
- both groups activate the MAP kinase pathway
what is the location of M1 muscarinic receptors?
- autonomic ganglia (including intramural ganglia in stomach)
- glands: salivary, lacrimal, etc
- cerebral cortex
what is the location of M2 muscarinic receptors?
heart: atria
CNS: widely distributed
what is the location of M3 muscarinic receptors?
exocrine glands: gastric (acid-secreting parietal cells), salivary etc
smooth muscle: GI tract, eye, airways, bladder
blood vessels: endothelium
what is the location of M4 muscarinic receptors?
CNS
what is the location of M5 muscarinic receptors?
CNS: very localised expression in substantia nigra
salivary glands and iris/ciliary muscle
what is the cellular response to M1 activation?
- increased IP3, DAG depolarisation
- excitation (slow epsp)
- decreased K+ conductance
what is the cellular response to M2 activation?
- decreased cAMP
- inhibition
- decreased Ca2+ conductance
- increased K+ conductance
what is the cellular response to M3 activation?
- increased IP3
- stimulation
- increased [Ca2+]
what is the cellular response to m4 activation?
- decreased cAMP
- inhibition
what is the cellular response to M5 activation?
- increased IP3
- excitation
what is the functional response to M1 activation?
- CNS excitation
- improved cognition?
- gastric secretion
what is the functional response to M2 activation?
- cardiac inhibition
- neural inhibition
- central muscarinic effects
what is the functional response to M3 activation?
- gastric, salivary secretion
- GI smooth muscle contraction
- ocular accommodation
- vasodilation
what is the functional response to M4 activation?
enhanced locomotion
what are non-selective M1 agonists?
- acetylcholine
- carbachol
- oxotremorine
- pilocarpine
- bethanechol
what are selective agonists for M1 and M3?
M1: McNA343
M3: Cavimeline
what are non-selective antagonists for M1?
- atropine
- dicycloverine
- tolterodine
- oxybutynin
- ipratropium
what are selective agonists for M1, M2, M3 and M4?
M1: Pirenzepine and Mamba toxin MT7
M2: Gallamine
M3: Darifenacin
M4: Mamba toxin MT3
what is Cevimeline used for?
- selective M3 agonist
- used to improve salivary and lacrimal secretion in Sjogren’s syndrome
what is Sjogren’s syndrome?
an autoimmune disorder characterised by dryness of the mouth and eyes
what has pirenzepine been used for?
peptic ulcer disease
what is darifenacin used for?
urinary incontinence in adults with detrusor muscle instability
what is Gallamine used for?
was used as a neuromuscular blocking drug
what are the classic muscarinic antagonists?
atropine and hyoscine
what is the process of acetylcholine synthesis?
- choline is taken up into the nerve terminal by a specific transporter
- free choline within the nerve terminal is acetylated by a cytosolic enzyme, choline acetyltransferase (CAT), which transfers the acetyl group from acetyl-CoA
what is the concentration of choline in the blood and body fluids? how does this change as it gets closer to cholinergic nerve terminals?
- normally 10 micromol/l
in immediate vicinity of - cholinergic nerve terminals: 1 mmol/l
- more than 50% of hydrolysed released ACh is recaptured by the nerve terminals
what is the rate limiting process in ACh synthesis?
choline transport, which is determined by the extracellular choline concentration and is linked to the rate at which ACh is released
what does ACh act on?
- acts postsynaptically on a nicotinic ACh receptor controlling a cation channel
- acts presynaptically on a nicotinic receptor that acts to facilitate ACh release during sustained synaptic activity
what does acetylcholinesterase break ACh down into?
choline and acetate
what inhibits AChE?
anticholinesterases e.g. neostigmine
what does inhibition of AChE cause?
- amount of free ACh and rate of leakage of ACh via the choline carrier is increased
how is choline transferred back into the presynaptic nerve?
choline carrier, which is coupled with ACh leaving
