Tolerance & Dependence

Question 1

What are the main causes of opioid deaths in UK?

Answer 1

All opioids deaths increasing (after period of decline) due to increasing purity of heroin; (before - purity of heroin was reducing)

Majority of deaths = heroin- largely metabolised to morphine so quickly that pick up morphine in blood results, but likely to be heroin

Methadone deaths have not increased (may have taken meth + heroin together)

Tramadol/oxycodone = prescription opioids, deaths are not increasing
* Fentanyls: not a big problem in UK so still quite small

Question 2

What are the main causes of opioid deaths in US?

Answer 2

Increase in heroin deaths (prescription opioids prescribed more widely in US, people switch back from oxycodone/dihydrocodone to heroin)

*Prescription opioids big cause of death; not prescribed as widely in UK/more controlled: deaths due to FENTANYLS (opioid for breakthrough cancer pain i.e. with sudden movement it breaks through the medication levels that keeps pain controlled)

Fentanyl has become an illicit drug, largely non-prescription; propyl-fentanyl/butyl-fentanyl, about 15 analogues that can be cut into heroin
*only pure fentanyl is prescribed

Question 3

Why is physical dependence unlikely to be main driver of addiction?

Answer 3

In modern day, lots of therapies to reduce intensity of withdrawal symptoms e.g. alpha-2 agonist; about 80% of people relapse; means that psychological dependence is bigger driver for the drug taking

Question 4

What is tolerance?

Answer 4

Loss of responsiveness over hours to days

*desensitisation may also be involved in this

Question 5

What is desensitisation?

Answer 5

Loss of receptor responsiveness over seconds to minutes

Question 6

What is tachyphylaxis?

Answer 6

Loss of responsiveness but without any specific mechanism (could be receptor desensitisation, or second messenger system that changes despite receptor functional)

Slightly old-fashioned term

Question 7

How does prolonged receptor activation cause desensitisation at the molecular/cellular level (3 things)?

Answer 7

1. Receptor uncoupling (µ-opioid receptors is GPCR so can uncouple from effector mechanism)
2. Receptor internalisation (internalised rec. may still signal, but probably only as minor component of the receptor function)
3. Receptor downregulation (internalised receptor sometimes degraded by lysosome)

Note: internalisation is NOT what causes desensitisation, receptor is DESENSITISED when in the plasma membrane (if desensitised then internalised, gives long term loss of receptors + contributes to loss of response, but INITIAL loss of response is simply due to arrestin binding)

Question 8

How does receptor uncoupling + internalisation occur?

Answer 8

If GPCR activated for prolonged period, becomes phosphorylated through G protein receptor kinases (GRKs), or possibly other kinases

If GRK: arrestin binds when MOR is phosphorylated. As arrestin bound, MOR can no longer interact with G-protein (uncoupling).

One possible result of this is internalisation (another possibility is arrestin signalling…)

Question 9

What happens once the u-opioid receptor is internalised?

Answer 9

Two options:

1. Sent to lysosome + degraded
   2: Dephosphorylated + recycled back to plasma membrane (this is not a slow/unimportant process - its very important!)

Question 10

How do µ-opioid receptor (MOR) splice variants affect desensitisation? Evidence for this?

Answer 10

17-18 splice variants exists (MOR1A + MOR1B = most common). MOR1B desensitises LESS rapidly than MOR1

Cells expressing MOR1/MOR1B pre-incubated with DAMGO for 2 hours: washed DAMGO out then applied another dose - response goes down (desensitises) compared to cells that were not incubated. MOR1B desensitised less rapidly than MOR1.

*Hypothesis: MOR1B doesn’t have all the phosphorylation sites that GRKs act on, so less phosphorylation occurs?

Question 11

What is DAMGO?

Answer 11

Endogenous peptide analogue- acts like morphine, but 1) higher intrinsic agonist efficacy 2) causes more arrestin binding
3) causes more receptor internalisation

i.e. morphine does not activate GRK-arrestin process well..

Question 12

How do µ-opioid receptor (MOR) splice variants affect internalisation? Evidence for this?

Answer 12

MOR1B internalises faster than MOR1, but desensitises more slowly.

Pre-labelled receptors with antibody, then applied DAMGO. Able to visualise receptors leaving membrane, but can’t see them reappearing. *MOR1B internalises faster, but recycled faster. MOR1B therefore shows less desensitisation because some receptors have been internalised and recycled back to membrane (functional).

MOR1: desensitised on membrane + waiting to be internalised, or internalised but still waiting to be recycled.

Arrestin causes desensitisation, whereas Internalisation & recycling REDUCES desensitisation (internalisation alone, or internalisation + degradation, would enhance desensitisation, but with MOR - rapidly moves through endoscopes and recycled).

Question 13

Why is the GRK-arrestin process of desensitisation unlikely to explain morphine tolerance?

Answer 13

Morphine does not activate GRK-arrestin process well.. (see DAMGO comparison)

BUT morphine (metabolite of heroin) does induce tolerance! (animal experiments have shown this, also evidence from addicts)

Question 14

How does morphine cause tolerance?

Answer 14

1. Continuous signalling ‘recruits’ a mechanism responsible for tolerance (‘rave’ hypothesis: receptor keeps on signalling when morphine used (as not desensitised) - this signalling switches on another mechanism that causes tolerance i.e. tolerance mechanism is separate from receptor desensitisation)
   - BUT this mechanism has not been found, also thinks based on partial agonists without accounting for the fact that partial agonists have antagonist effects
2. Another mechanism is responsible for desensitisation AND tolerance

Question 15

What did MacPherson et al (2010) show about arrestin recruitment?

Answer 15

Arrestin recruitment correlates with operational efficacy for GTPƔS binding

Morphine has low efficacy to activate G proteins and to activate arrestin (therefore will have lower maximum response)

DAMGO = highest efficacy agonist we have for G-protein activation, also one of highest efficacy for causing arrestin translocation

Question 16

How do high efficacy agonists cause MOP desensitisation?

Answer 16

e.g. DAMGO + Met Enk

In addition to GRKs / arrestin, range of kinases interact with MOR

DAMGO: GRK-2 + GRK-3 phosphorylate MOR, causing arrestin binding and desensitisation

Also evidence that ERKs may phosphorylate the receptor (but not as strong evidence) - Williams et al 2013

Question 17

• Williams 2013 review article: question mark for ERK role

Answer 17

?? review this

Question 18

??

Answer 18

Best experiments to study desensitisation are on real neurons (more physiologically relevant than HEK cells etc)

Most studied = neurons from locus coeruleus

*Because opioid receptors are Gi/Go coupled, activation causes potassium channels to open (potassium efflux) - therefore to study MOR function, can study potassium current in response to µ-agonists

Question 19

How do high efficacy agonists desensitise opioid receptors in locus coeruleus neurons? (first experiment)

Answer 19

Bailey et al

Hypothesis: high efficacy agonists will cause GRK involvement

Inject dominant negative GRK2 mutant: neurons of intact animal over-express non-functional GRK which blocks activity of functional GRK in neurons of intact animal

When DAMGO added, outward current response decays with time (because receptors desensitised), but in GKR2 mutant cells: less desensitisation.

Also less desensitisation in GKR3 KO (GRK2 KO is embryonically lethal).

Good evidence that GRK2 or 3 is involved in desensitisation

Question 20

How do high efficacy agonists desensitise opioid receptors in locus coeruleus neurons? (2nd experiment)

Answer 20

Bailey et al

Compound 101: developed as inhibitor to GRK2/3

Met-Enkephalin activates receptors, then decay due to desensitisation

If add compound 101, don’t get decay, less desensitisation

*Good evidence that for high efficacy MOR agonists - GRKs and arrestin involved in desensitisation

Question 21

What did Dang et al show?

Answer 21

Locus coeruleus neurons

Desensitisation to Met-Enkephalin, added inhibitory peptides to pipette, allowed them to diffuse into neuron

GRK2 inhibitor: does not change desensitisation. ERK1/2 inhibitor: may be a little bit reduced

ERK1/2 and GRK2 inhibitors together: marked reduction in desensitisation

Suggests 2 processes: GRK2/3 and ERK1/2

Question 22

Why do Dang + Bailey disagree?

Answer 22

Bailey experiments suggests can inhibit desensitisation with a GRK inhibitor (dominant negative mutant + compound 101)

• doesn’t affect ERK so how does this make sense? Bailey et al have also used ERK inhibitors and found no change in desensitisation

Question 23

Williams (2013) review aims to find common ground between the two theories, explanation for the different results
* may argue good evidence for GRK, some evidence for ERK, why the labs are different unknown

Answer 23

add reading

Question 24

What did Bailey et al show about GABAergic neurons?

Answer 24

Cell body patch clamp: GABAergic neurons on the VTA: involved in reward process

1. Applied DAMGO to post-synaptic VTA cell body: recorded K current, then desensitises (MORs on nerve terminals, inhibit NT release)
2. Recorded from pre-synaptic cell body (no µ receptors therefore would not have K current). When DAMGO applied - inhibitory synaptic currents that inhibit GABA release and now no desensitisation

Other groups studying brain neurons get same results: MORs on cell bodies desensitise, but the µ-opioid receptors on nerve terminals do not desensitise

Possible explanation: GRK + arrestin in cell body but nerve terminals do not contain either GRK/arrestin/both
*or receptors are different in nerve terminal, arrestin and GRK present but because receptors different they are not desensitised

*remember: base all theories on opioid tolerance/dependence on recordings from cell body and on desensitisation, but actually in lots of places the receptors don’t desensitise

Question 25

How was the role of PKC in desensitisation demonstrated?

Answer 25

Locus coeruleus neurons rat brain slices; - response to morphine, then activate / elevate the level of PKC activity (first by activating muscarinic Gq coupled receptors, second using PMA)

Increasing PKC activity = no change in efficacy / peak current, but desensitises quickly (effect blocked by chelerythrine which a PKC inhibitor, therefore PKC-dependent)

Limitation: gap between killing rat + performing experiment, neurons therefore in basal state, level of PKC activity may have quietened because the cells were not activated during the time interval
*Would mean GRK activity doesn’t go down - don’t need to ‘tickle’ system as get full response with experiment??

Question 26

What are the two different types of MOR desensitisation?

Answer 26

Met-Enkephalin largely GRK-mediated
Morphine largely PKC-mediated

*GRK-mediated: probably what happens in the cell body - but in the nerve terminal, probably no GRK - in nerve terminal DAMGO shouldn’t do anything

When GRK over-expressed, or endogenously high, may find morphine is both PKC and GRK dependent

In LC neurons, morphine doesn’t cause desensitisation, because there is no PKC
* but if increase activity of PKC start to see it

Question 27

Why can there be two different types of MOR desensitisation experimentally?

Answer 27

Maybe everyone is right - depends on the conditions of the system?

People often add GRK / different expression systems.

*Even if working on neurons in slice, lots depends on how you cut the slice e.g. can cut in a direction where have almost no dendrites, or with loads of dendrites - conditions not same across experiments even if all used locus coeruleus neurons

Question 28

What is the result of slowly releasing morphine over time? (slice evidence)

Answer 28

So far have seen desensitisation occurring within about 10 mins. In single neuron can also see development of tolerance.

Experiment: slowly release morphine daily - after 3 days animal killed + brain slices kept in morphine

Control animals: (emulsion with no morphine,): larger potassium current

Morphine for 3 days: maximum current is less (cells are becoming tolerant - less response to opioid)

PKC inhibitor added: reversal of tolerance (not 100% reversal)

Question 29

What is the result of slowly releasing morphine over time? (in vivo evidence)

Answer 29

Mice:

1. Naive, or pretreated with 4x morphine (SC) every 2 hours (8 hours). If pretreated, CRC shifts right (TOLERANCE - more morphine needed for same anti-nociceptive response - tail withdrawal to hot water). Repeat with PKC inhibitor to morphine pretreated: no longer tolerant
2. Repeat experimental procedure but use DAMGO (ICV as doesn’t cross BBB) instead of morphine. Again CRC shifts right with pretreatment (tolerance) but PKC inhibitor does NOT reverse tolerance. However, GRK2 inhibitor DOES reverse DAMGO tolerance.

Morphine tolerance = PKC dependent DAMGO tolerance = GRK dependent. (behavioural evidence).

• all low efficacy = PKC involvement
• high efficacy agonist = GRK / arrestin mechanisms

Question 30

For low efficacy agonists, PKC will reverse tolerance + desensitisation. How does it do this?

Answer 30

MOR sequence has various residues that are phosphorylated by PKC (especially 363) - common theory but evidence not that great?

*instead, may reduce desensitisation / tolerance by phosphorylating something else involved in signalling? e.g. the G-protein rather than the receptor?

Question 31

What evidence is there to suggest that tolerance to low efficacy agonist is caused by receptors other than MOR?

Answer 31

LC neurons in basal state in dish - must ↑ PKC activity to cause desensitisation. Maybe PKC activity low inside cell, but can be increased through several ways…?

1. Groups studying various receptors claim their antagonists reduce morphine tolerance. Maybe activity of these receptors involved? *all the receptors increase IC calcium..
2. Gq coupled receptors will also produce diacyl glycerol and therefore activate PKC (enhancing PKC activity)
3. NMDAR highly Ca-permeable + PKCs are highly calcium sensitive (more calcium = more conventional PKC isoforms active)

Theory: all receptors involved in tolerance for low efficacy agonists, therefore antagonist of these would reduce PKC activity and therefore reduce tolerance

Question 32

What did Levitt and Williams (2012) show?

Answer 32

Levitt & Williams did same experiment

Control, SP (neurokinin antagonist), PKC inhibitor)
*washing cells for an hour also reversed tolerance to about the same level of the PKC inhibitor

Therefore PKC component is rapidly reversible component
* don’t know what the other component is

Question 33

What did Cox et al (1975) show?

Answer 33

Made rats tolerant, then stopped giving opiate + measured analgesia:

Tolerance reversed very quickly (within 4 days) but not completely = there was a slow component (4-20 days)

With morphine - probably PKC component

With methadone - probably GRK/arrestin component)

Question 34

How has the role of JNK in morphine tolerance been demonstrated?

Answer 34

Tail flick assay: control vs GRK KO animals

1. Morphine (2 doses): both animals tolerant to 2nd dose (GRK not involved in morphine tolerance)
2. Fentanyl (2 doses): controls tolerant, but KOs had reversal of tolerance / no tolerance
3. JNK inhibitor to KOs: fentanyl tolerance is no longer reversed - and increasing dose of JNK inhibitor REVERSES morphine tolerance

Question 35

How has the role of JNK in morphine tolerance been demonstrated?

Answer 35

Tail flick assay: control vs GRK KO animals

1. Morphine (2 doses): both animals tolerant to 2nd dose (GRK not involved in morphine tolerance)
2. Fentanyl (2 doses): controls tolerant, but KOs had reversal of tolerance / no tolerance
3. JNK inhibitor to KOs: fentanyl tolerance remains, but increasing dose of JNK inhibitor REVERSES morphine tolerance

Conclusion: JNK causative role in morphine tolerance? but not in high efficacy agonists.

Question 36

Potential therapeutic implications for morphine tolerance

Answer 36

1. Enhance tolerance - reduce overdose? i.e. give to addicts after detox to reduce their risk of overdose (showed that phorbol esters enhance PKC activity + morphine tolerance, but cause cancer so cannot be used - need selective PKC activation within brain neurons)
2. Reduce tolerance - decrease methadone side effects
   
   • methadone = NMDA / potassium channel antagonist; if on maintenance therapy can be like ‘zombie’ e.g. could give potent GRK inhibitor with the methadone
   • not possible to patent GRK inhibitors but still being looked at
3. Polypharmacology
   
   • alcohol, pregabalin reduce morphine tolerance (enhance likelihood of overdose)

Question 37

Potential therapeutic implications for morphine tolerance?

Answer 37

1. Enhance tolerance to reduce overdose risk?
   particularly for addicts after detox (phorbol esters enhance PKC activity + morphine tolerance, but cause cancer - need selective PKC activation within brain neurons)
2. Reduce tolerance - decrease methadone side effects (methadone = NMDA / K channel antagonist; if on maintenance therapy could give potent GRK inhibitor with the methadone)
   * cannot patent GRK inhibitors- still being looked at
3. Polypharmacology: alcohol, pregabalin reduce morphine tolerance (and therefore increase risk of overdose)

Question 38

What is the enzyme expansion theory?

Answer 38

Historically, physical dependence + tolerance thought to be related, now thought to be separate phenomena.

Theory: there is basal level of enzyme activity + adding drug will inhibit enzyme in some way (in opioids - µ receptor Gi/Go coupled - decreases adenyl cyclase activity)

In response, cell synthesises new enzyme (adaptive phenomenon) - brings level of enzyme activity back up

*two types: inhibited and uninhibited - the uninhibited is newly synthesised

Then remove drug, and inhibited enzyme becomes active - overall enhancement of enzyme activity = withdrawal response

Tolerance + withdrawal thought to be reverse of the same phenomenon (tolerant by increasing activity of something, then when remove drug, reveal excess = withdrawal response

Question 39

What is the rebound phenomenon?

Answer 39

Still thought to be true, but not because of the development of tolerance

Brain neurons: adenyl cyclase activity relatively low, can inhibit with drug like morphine and produce tolerance

When withdraw morphine - huge increase in adenylyl cyclase activity - known as SUPERACTIVATION

*Nobody knows why super activation occurs - think receptors are desensitised because they are phosphorylated by PKC / GRK
(so not simply to do with adenyl cylase, but withdrawing opioid does produce superactivation)

Goldstein correct about withdrawal being a rebound phenomenon, but wrong in linking tolerance to rebound phenomenon

Question 40

What did Collier et al do to support the rebound theory?

Answer 40

If big increase in adenyl cyclase activity - increase cAMP in animals should produce withdrawal response

Gave theophylline (phosphodiesterase inhibitor - stops cAMP breakdown)

Withdrawal symptoms in rats e.g. jumping
- theophylline with no morphine produced the symptoms! i.e. increase in cAMP

Question 41

What have Bagley et al shown?

Answer 41

Normally, opiate receptor on nerve terminal would inhibit transmitter release (recorded from neurons + measured NT release - opiates acutely inhibit transmitter release)

If give chronic morphine (bathing fluid), then give naloxone - everything much more excitable

Hypothesised: in nerve terminals cAMP increases (Goldstein theory) and this would create increase in PKA

Increase in PKA would change release mechanism to give more NT release, or would phosphorylate GABA transporter (GABA transporter = sodium influx = more NT release)

*Has done more papers showing that PKA inhibitors prevent this increase in excitability and NT release - fits with idea of superactivation - shows that increase in cAMP makes everything more excitable by phosphorylating the transporter