Opioids and Opioid Receptors:

Question 1

What is the definition of an opioid?

Answer 1

naturally-occurring, synthetic or semi-synthetic compounds which act at opioid receptors

Question 2

What are these naturally-occurring compounds also called?

Answer 2

Opiates - they are derived from the resin of the opium poppy

Question 3

Give 3 examples of opioids

Answer 3

Morphine
Codeine
Thebaine

Question 4

What are morphine esters derived from? What do they include?

Answer 4

Morphine esters are derived from opium.

They include morphine prodrugs such as diacetylmorphine - AKA heroin

Question 5

What do semi-synthetic opioids include? What are they derived from?

Answer 5

o Semi-synthetic opioids (derived from opium or morphine esters) include buprenorphine and oxycodone

Question 6

What do synthetic opioids include?

Answer 6

o Synthetic opioids include fentanyl, pethidine, methadone, and tramadol

Question 7

What do endogenous opioids include?

Answer 7

Enkephalins and endorphins

Question 8

What are the effects of opioids? Is their abuse potential low or high?

Answer 8

Effects:
o Surge of pleasurable sensation (“rush”)
o Reduced stress and anxiety
o Sedation
o Impaired mental function
o Cardiac and respiratory function slows – can be life-threatening
o Reduced pain – physical and psychological

Opiates (in the form of opium) have been used and abused since antiquity – evidence suggests the opium poppy was cultivated as far back as 3400BC
• Opioids (includes opiates) have a very high abuse potential

Question 9

Is diacetylmorphine an active drug?

Answer 9

No, but it is rapidly metablised into active drugs

Question 10

What is diacetylmorphine metabolised into? Which of them are active compounds?

Answer 10

Diacetylmorphine –> 6-monoacetylmorphine (active) + 3-monoacetylmorphine (inactive) –> morphine (active)

Question 11

What gives diacetylmorphine its lipid solubility? What are the implications of this?

Answer 11

o The 2 acetyl groups make the compound highly lipid soluble – it can cross the BBB much more easily than morphine

Question 12

Which gene encodes proenkephalin?

Answer 12

The PENK gene

Question 13

How many forms of enkephalin are there? What are they called and why?

Answer 13

2 forms:
[Met]enkephalin - AA sequence is Tyr-Gly-Gly-Phe-Met
[Leu]enkephalin - AA sequence is Tyr-Gly-Gly-Phe-Leu

Question 14

What does xM mean?

Answer 14

Extended [met]enkephalin

Question 15

What is the ratio of the amount of [met]enkephalin to [leu]enkephalin produced by the body?

Answer 15

4:1 ratio

Question 16

Which gene is prodynorphin encoded by?

Answer 16

PDYN

Question 17

How many isoforms of dynorphin are there? Name 5 of these.

Answer 17

7 isoforms:

	α-neo-endorphin
	β-neo-endorphin
	Big dynorphin A
	Dynorphin A (various sizes)
	Leumorphin
	Dynorphin B
	Leu-enkephalin

Question 18

Which gene encodes beta-endorphin?

Answer 18

POMC gene, encodes proopiomelanocortin. Beta-endorphin is the only opioid produced by POMC

Question 19

Which N-terminal sequence is encoded by all endogenous opioids?

Answer 19

either [Met]- or [Leu]-enkephalin

Question 20

Name the four types of opioid receptors, their endogenous agonists, their signalling mechanism, and the downstream effects of this mechanism

Answer 20

δ – agonised by β-endorphin, [Leu]enkephalin and [Met]enkephalin
κ – agonised by big dynorphin and dynorphin A
μ – agonised by β-endorphin, [Leu]enkephalin and [Met]enkephalin
NOP – agonised by nociceptin/orphanin FQ

All signal via Gi/o signalling - many downstream messengers and effects:
Affects K+ and Ca2+ channels
Desensitisation via interactions with GRK, βarr, PKC, PKA
Endocytosis via interactions with AP2, PLD2, Dyn, βarr, clathrin
Interacts with ERK, JNK, and βarr for G-protein-independent signalling

Question 21

List 4 uses of opioid receptor agonists and state what they are used to treat

Answer 21

Agonists – used for pain management:

1. Acute pain management
2. Pain management in palliative care
3. Long-term pain management
4. Supplement to general anaesthesia
5. Primary anaesthesia

Question 22

What are opioid receptor antagonists used to treat? Name one.

Answer 22

Antagonists – treat opioid overdose:

o Naloxone – non-selective competitive opioid receptor antagonist

Question 23

Briefly describe the sensory transduction pathways for noxious and non-noxious stimuli.

Answer 23

Non-noxious and mechanical stimuli are transduced into action potentials within Aβ-fibres of the PNS; these are then relayed through the dorsal root ganglion to the CNS (thalamus and primary sensory cortex)

Noxious mechanical stimuli are transduced into APs within Aδ-fibres of the PNS; these are then relayed through the dorsal root ganglion into a motor neuron (to effect an immediate response) and to the CNS (thalamus and primary sensory cortex)

Noxious heat and chemical stimuli are transduced into APs within C-fibres of the PNS; these are then relayed through the dorsal root ganglion into a motor neuron and to the CNS (thalamus and primary sensory cortex)

Question 24

What is rate coding?

Answer 24

Coding of intensity – weak stimuli have APs firing less frequently
o Strong stimuli have APs firing more frequently
o APs don’t get bigger; they become more frequent as the intensity of a stimulus increases
• At the spinal cord, the AP from the dorsal root ganglia/nodose ganglia/trigeminal ganglia triggers neurotransmitter release to pass the signal along to the CNS

Question 25

How do opioids suppress pain?

Answer 25

Opioids do not block the neurotransmitter receptor

μ-opioid receptors are Gi/o-coupled; therefore these negatively couple (or suppress) the activity of Ca2+-channels (voltage dependent and voltage independent) –> reduced synaptic transmission

Question 26

Explain, with the aid of a diagram, descending control of nociception in the spinal cord.

Answer 26

There is a descending 5-HT projection (in the dorsolateral funiculus) from the Raphe nuclei which stimulates GABAergic and enkephalinergic interneurons, causing these to release GABA and enkephalins (respectively). The 5-HT projection also inhibits the spinal projection neuron via a 5-HT1AR.

GABAergic interneurons inhibit the spinal projection neuron by releasing GABA.

Enkephalinergic neurons inhibit spinal projection cells via opioid receptors (coupled to K+ channels –> cell hyperpolarisation ∴ suppressing APs).
Enkephalins also depress Glu release from the primary afferent neuron (bringing pain signals from the PNS).

Primary afferent neuron normally excites the spinal projection neuron, causing pain detection. Therefore by blocking the mechanisms listed, we can control nociception.
(Diagram in lecture notes)

Question 27

What are the major sites of analgesic opioid actions? Are they pre- or post-synaptic?

Answer 27

1. Periaqueductal grey: opioid receptor activation causes activation of periaqueductal grey projection neurons, by inhibiting the ongoing synaptic inhibition they normally receive
2. Presynaptic terminals of primary afferent nociceptors: this depresses the release of glutamate and therefore depresses synaptic excitation
3. Post-synaptically in spinal cord projection neurons – this inhibits activity in the spinothalamic tract by activating K+ channels –> hyperpolarisation of the neuron

Question 28

List 5 situations wherein pain sensation may be reduced.

Inhibition of which pathways may play a role in this?

Answer 28

o Fakirs – Sufi Muslims who have taken vows of poverty and worship, renouncing all relations and possessions; they’re self-sufficient and possess only the spiritual need for Allah. Renowned for lying on nail-beds and feeling no pain
o Battlefields or accidents – people not noticing injuries, or overcoming normal human limits such as mothers lifting cars to get their children out
o Redirection of attention – focussing on something else
o Hypnosis
o Altered emotions/motivation -
o Acute injuries – stimulating the skin near the site of the injury

Descending inhibition of the periaqueductal grey, presynaptic terminals of primary afferent nociceptors, and postsynaptically in spinal cord projection neurons

Question 29

List 6 side effects of taking opioids

Answer 29

•	Addiction
o	Often --> abuse
•	Tolerance
o	Occurs on a clinical, behavioural, and cellular level
•	Sedation
•	Itching
•	Dry mouth
•	Dizziness
•	Nausea
•	Vomiting
•	Respiratory depression
•	Constipation

Question 30

Why is constipation a health economics issue? Present a potential solution.

Answer 30

Health economics issue – patients who’ve undergone major surgery are usually treated with opioids for post-surgical pain relief –> constipation.

Patients can’t be discharged until they’ve defecated – opioid treatment –> bed-blocking

This occurs because opioids reduce normal neurotransmitter release in the GI tract. Normally, GI motility relies on a fine balance between excitatory and inhibitory neurotransmitters released by myenteric neurons acting on smooth muscle

Taking opioids –> abnormal coordination of smooth muscle contraction and relaxation –> increased muscle tone in the small and large intestinal circular muscle; causing constipation

Solution:

Peripherally-restricted μ-opioid antagonists:

Alvimopan

Polar molecule ∴ low lipid solubility so can’t cross BBB well

In oral formulations it’s very poorly-absorbed –> concentrating effect in GI tract

Question 31

Explain the molecular mechanisms which may contribute to opioid tolerance. Use a diagram.

Answer 31

Initially (0-0.1s) – prolonged agonist binding desensitises GPCR (G-proteins uncoupled)

Long term (1s-hours) – c-terminus sites are phosphorylated –> β-arrestin2 binding, signals to other clathrin-coated mechanisms –> endocytosis of receptor, so no longer activated by exogenous agonists

Reversal of these processes via receptor cycling reduces tolerance

Question 32

Describe a piece of evidence in favour of beta-arrestin2 being involved in tolerance

Answer 32

β-arrestin2 KO mice don’t develop an anti-nociceptive morphine tolerance – suggests β-arrestin2 is required for tolerance development.
Tested via hot-plate response latency (plate at 56°C); tested maximum possible effect (MPE).

%MPE = 100% x ((drug response time-basal response time))/((maximum test duration-basal response time))
Maximum test duration is usually 30s to avoid tissue damage

Question 33

Why are synthetic opioids such as fentanyl more potent than natural opioids in vivo, despite similar binding and potency in vitro? How does this contribute to the lethality of fentanyl?

Answer 33

Fentanyl has greater potency in vivo but similar binding and potency in vitro compared to heroin

Receptor binding Ki = 1.35nm (morphine) 1.17nm (fentanyl)
[35S]GTPγS binding (functional) logEC50 = -6.4 (morphine) -6.8 (fentanyl)

Difference in potency is due to fentanyl being ~3000x more lipophilic than morphine, so fentanyl can cross the BBB more easily and therefore produce more potent (and potentially fatal) effects at lower doses
Log P 1.07 (morphine) 4.28 (fentanyl)

More drug in the CNS –> more μ-opioid receptor activation –> greater CNS effects (e.g. greater respiratory depression via μ-opioid receptors in respiratory areas in the brain stem), potentially death