Tolerance & Dependence Flashcards

Question

How was the role of PKC in desensitisation demonstrated?

Answer 1

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??

Answer 2

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

Answer 3

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

Answer 4

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)

Answer 5

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

Answer 6

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?

Answer 7

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

Answer 8

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

Answer 9

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)

Answer 10

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

Answer 11

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.

Answer 12

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)

Answer 13

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)

Answer 14

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

Answer 15

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

Answer 16

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

Answer 17

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

Tolerance & Dependence Flashcards

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