Local Anaesthetics Flashcards
What are piezo receptors?
Piezo proteins constitute a family of excitatory ion channels directly gated by mechanical forces. These ion channels are involved in cell mechanotransduction — the conversion of mechanical forces into biological signals
Describe TRPV1 channels
Vannilinoids; non selective cation channels that respond to thermal stimuli
Sodium moves via conc gradient to depolarize the membrane potential
What are the sodium and potassium equilibriums respectively?
Sodium; 60 mV (depolarization)
Potassium; -80mV (hyperpolarization)
Describe the different phases of the action potential
Resting state (A); primary ion channel open is the potassium leak channel (constitutive channels)
Depolarizing phase (B); voltage activated sodium channels open, allows sodium influx. Initially K+ channels are closed, they activate more slowly
Repolarizing phase (C); Na+ become inactive, K+ opens allowing for K+ efflux
Undershoot (K); sodium channels not deactivated, K+ open. Membrane potential becomes hyperpolarized
Back to the resting state (A)
What is the threshold for an AP?
-50mV; IF the sodium channels are in the resting state
All or none
What is the difference between inactivation and deactivation?
Inactivation; channel otherwise in open state, but has become inactivated (h gate closes)
Deactivation; ion channel closes (M gate)
What do C fibres respond to?
Polymodal; temp, mechanical, inflammatory
Inflammatory mediators; bradykinin, serotonin, prostaglandin and histamine)
Why is there a difference in conduction between unmyelinated and myelinated axons?
In unmyelinated axons; the whole membrane is required to become depolarized for conduction of the action potential
Myelinated axons; Schwann cells allow for insulation, action potentials can undergo salutatory conduction whereby they jump from one node of Ranvier to the next
What mediated fast and slow pain?
Fast pain; A-delta (blocked by LA)
Slow/ second pain; C-fibres (blocked by NSAIDs and LAs)
What is the site of action of LA?
Voltage activated sodium channels
Which aspects of the voltage activated sodium channel acts as voltage sensors?
4th membrane spanning domain of 1,2,3 and 4th repeat
6 spanning transmembrane proteins
Voltage sensor on 4th domain (+ve charged amino acids that respond to membrane potential)
Re-entrant loop between 5 and 6 - lines the ion channel pore. Enables the ion channel to attract +ve Na+ ions
H-gate (inactivation part)
N and C terminal are intracellular
Are there different types of sodium channels?
Yes; SNC 1-11
TTX sensitive isoforms generally activate at more depolarized potentials compared to TTX resistant forms
There are 4 beta subunit genes (b1-4)
NaV 1.7, 1.8, 1.9 are nociceptive
NaV 1.3 are also involved with nociceptive transmission
What is the difference between how TTX and LA block these voltage activated Na+ channels
TTX extracellularly blocks the channel. Independent of inactivation
LA gains access to the channel through the intracellular environment or lipid bilayer and trans versing the channel. Use dependent block; the channel must be activated for LA to block. Highest affinity when it enters the inactivated state, stabilises the channel in the inactive state
What is the difference between an ester and an amide LA?
Lidocaine; amide
Cocaine; ester
All LA have an aromatic portion (benzene ring) - allows for lipid solubility and hydrophobicity
Intermediate chain; longer the chain, the more lipid soluble the LA
Amine portion; enables charge at appropriate pH
Amides; contain an amide link (nitrogen and hydrogen)
Ester link; carbon to oxygen link
What LAs are esters and amides
Ester; cocaine, procaine, tetracaine, benzocaine
Amides; lidocaine, mepivacaine, bupivacaine, etidocaine, prilocaine, ropivacaine
Why does it matter whether a LA is an ester or an amide?
Dictates the route through which the LA can become metabolised
Esters; Short duration (metabolized by plasma and liver pseudocholinesterase's) Allergies; most likely for esters - PABA metabolites produced by metabolism through pseudocholinesterase's
Amides;
Longer duration of action; metabolised in liver CYP3A4 p450 enzyme
When administered correctly, LA do not have behavioural effects . If enters systemic circulation; visual or taste symptoms (systemic toxicity). LA in CNS; block lots of ion channels causing seizures - treat with BZD
What does it mean to be amphipathic?
A molecule that is both hydrophilic and hydrophobic
Are LA amphipathic?
Yes
Contain an amine group (can be uncharged or charged)
Uncharged; basic
Charged; proton acceptor; cationic form. At physiological pH, the cationic form cannot enter the cell to block the sodium channel, limits the level of LA that can enter the cells
How can the ratio of charged and uncharged form of LA be calculated?
Henderson-Hasselbach Equation
Log (cationic form/ uncharged form) = pKa -pH
What is the normal range of pKa of LA?
7.7-9.1
Average of 8
At physiologic conditions; what is the ratio of cationic form to uncharged (basic) form?
10x more cationic (cannot enter the cell)
At any given dose, only 1/10th of the LA has access to the inside of the cell
Is the pH always 7 within the human body?
No; infected tissue has a lower pH, leading to a more charged LA
This results in even less anaesthetics able to cross the cell membrane
What must LA cross to exert its effects?
Nerve sheath - into extracellular space
Nerve membrane - into axoplasm
Uncharged will cross the membrane, however the charged form will block the channel
The LA must be amphipathic to cross the membrane, and then exert its actions at the voltage activated sodium channels
LA can also interact with K+ channels, and GABA-A receptor
Are all axons equally susceptible to blockade?
No; they are all sensitive to blockade
However, the most sensitive are unmyelinated fibres. This is because they have a smaller diameter
However, the myelinated fibres are blocked more easily that unmyelinated fibres of the same diameter. LA can block the node of Ranvier.
The smaller the diameter of the axon; the higher degree of blockade via LA. Smaller diameters have a high electrical resistance, and therefore blockade has a tendency to prevent the AP from spreading
Wider diameters; have a lower resistance; the AP can still spread due to conduction down the fibre
How can LA cause arrhythmias?
The can interact with cardiac NaV 1.5 Na+ channel, modulating its function
What is the link between pain and NaV1.7?
Residues in NaV1.7 can be mutates (amino acid sub)
Inherited erythromelalgia (burning sensation in extremities); mutations that affect amino acids within NaV1.7
Paroxysmal extreme pain disorder
Congenital insensitivity to pain
Gain of function; NaV1.7 more likely to be activated
Loss of function; NaV1.7 has reduced activity = insensitivity to pain
Where is NaV1.7 expressed?
TTX sensitive
Expressed selectively in DRG, particularly in nociceptive cells
What is the role of NaV1.7 receptor?
Sets the gain for pain in nociceptors
NaV1.7 KO = increased mechanical and thermal pain thresholds + reduced inflammatory pain responses
What does a gain of function mutation in NaV1.7 result in?
L858H, N395K
Severe burning pain and redness in extremities in response to mild thermal stimuli (erythromelalgia)
What does a loss of function of NaV1.7 result in?
Unable to feel pain (acute or chronic)
Sub-type specific NaV1.7 blockers therefore can act as novel analgesics
Would individuals with a gain of function NaV 1.7 mutation show any differences with response to lidocaine?
Yes; show a reduced sensitivity
Lower efficacy as analgesic in these individuals
Describe the paper; defining the functional role of NaV 1.7 human nocicpetion
Patients with CIP due to NaV1.7 mutations have a functional absence of nociceptors
Lack of nociceptive drive is reflected in a reduced cortical response to capsaicin
Patient iPSC nociceptors show reduced excitability, especially to ramp stimuli
Gene-edited iPSC nociceptors represent a valuable analgesic drug screening platform
Complete insensitivity to mechanical pain threshold, and thermal sensory threshold
Histamine and mustard oil flare response on the forearm of a CIP participant
Action potential in C-fibres gives rise to flare response. The flare response is present in the CIP participant.
Skin biopsy of CIP vs healthy.
CIP = absence of intraepidermal nerve fibres . Suggests the mutations give rise to changes in the structure and functional of the somatosensory system
Can you evoke action potentials in neurons from CIP patients?
Yes; In CIP patients; more current is required to induce an AP (rheobase)
Less APs produced
Is there a role for endogenous opioids in insensitivity to pain (mutations in NaV 1.7)?
Minett et al; 2015
The pain deficit seen in these individuals is due to increased endogenous opioid signalling
Administration of naloxone can restore pain sensitivity in these patients
NaV1.7 K/O mice; enhanced expression of metenkephalin within DRG
We report that Nav1.7 deletion leads to increased transcription of Penk messenger RNA and higher levels of enkephalins in sensory neurons.
The analgesia associated with loss of Nav1.7 in both mice and humans is substantially reversed by the opioid antagonist naloxone.
Thus, Nav1.7 deletion increases endogenous opioid-dependent analgesia as well as diminishing peripheral nociceptive drive in pain states
Describe the grasshopper mouse
Evolved to withstand venom of bark scorpion via introduction of mutations into NaV1.8 = and therefore pain transmission
Grasshopper mouse is immune to the pain of the venom
Describe the bark scorpion venom pain mechanisms
Rowe et al; 2013
Binds to voltage gated sodium channel NaV1.7
Can also bind to NaV1.8 sodium channel; inactivates this channel and in so doing produced the other aspects of the venom to cause pain
Does bark scorpion venom cause pain in grasshopper mouse? (rowe et al 2013)
No:
Paw licking behaviour is less when injected with the venom in comparison with other species of mice
For comparison, these mice were also injected with an irritant; and the grasshopper and musculus species both showed increased pain licking
Therefore, this insensitivity to the venom is not due to a global reduction in pain transmission
Are there differences in electrophysiological recordings in grasshopper mice?
Looked at the relationship between depolarization and current (voltage clamp from single DRG neurons) and the response to venom
Reduction in current amplitude in grasshopper mice in comparison with the other mice - less likely to fire an AP = no pain transmission
Reduction in maximal current, concentration depend reduction in current amplitude
What area of NaV1.8 in grasshopper mouse is critical for sensitivity to bark scorpion venom?
A single amino acid residue is substituted
E (glutamic acid) to Q (glutamine) at 862 confers venom sensitivity in grasshopper mice
Do LA just target the nociceptive fibres?
No; other channels involved with important activities - such as numbness and motor control
LA can reduce motor activity - problematic during recovery
Target nociceptive neurons specifically; ultrasound guided anaesthesia to delivery anaesthetic directly onto the nerve
Binshtoc et al; 2007
Targeting nociception with lidocaine derivative that is charged (LA are amphipathic and the form off the LA as cationic or basic is based off the pH and the pKA)
Charged lidocaine (Qx-314) can be targeted to nociceptive neurons using TRPV1 channels (capsaicin, able to pass large organic cations (+ve charged) molecules)
It cannot cross the cell membrane (as it cannot adapt a basic form)
Describe the impact of QX-314 on sodium currents in DRG neurons
When co-applied with capsaicin; QX-314 inhibits sodium currents
Therefore, QX-314 can gain entry to the DRG neuron via TRPV1 channel to selectively block sodium channels
Can this action of QX-314 to inhibit sodium currents when co-applied with capsaicin?
Via von frey filaments; score when the animal moves the paw in relation to mechanical stimulus. This mechanical threshold was much higher in capsaicin and QX-314 animals
The same was true for thermal threshold
Injection of QX-314 followed by capsaicin adjacent to sciatic nerve anaesthetized animals to noxious mechanical and thermal stimuli without producing any motor deficits
Describe the use of antibodies that are anti-nociceptive (novel analgesia)
Lee et al; 2014
Ab can be developed against specific amino acid sequences within NaV1.7 to incapacitate its ability to activate
Anti-ouch and anti-itch Ab
These antibodies can reduce the amplitude of the sodium channel conductance - this is specific to NaV1.7 - inhibited in a potent, dose dependent concentration by this monoclonal Ab
This Ab reduced inflammatory and neuropathic pain without affecting motor coordination and balance - predominantly in phase 2 of pain (10-45 mins after formalin) with no effect on motor skills
IV and intrathecal Ab; reduction in pain behaviour
Describe the black mamba venom toxin (mambalgin)
Diochot et al; 2012
Anaesthetizes prey
Isolated; venom toxin has mambalgin which is a large peptide
Inhibits pH sensitive acid channels (ASIC)
Mambalgin is analgesic however it doesn’t result in resp depression
This suggests that ASIC channels may be good targets for new analgesics
