The Neuromuscular Junction

• Autonomic nervous system:

  • Involuntary (smooth muscle, cardiac, exocrine and some endocrine)
  • Regulated by brain stem centres
• Somatic nervous system:

  • Voluntary body movements
  • Regulated by corticospinal tracts and spinal reflexes
• Nicotinic receptors can be present on both the preganglionic (sympathetic) and postganglionic (parasympathetic) neuron. There is no section between somatic neurons -the tissue receptor is nicotinic.

  
• Acetylcholine synthesis:

  Pyruvate -> Acetyl-CoA + choline -> acetylcholine -> acetate + choline

  (catalysed by pyruvate dehydrogenase, choline acetyltransferase and acetylcholinesterase)
• The nicotinic receptors are N1/Nm or N2/Nn (muscular or neuronal). These are ligand-gated, requiring the binding of two ACh molecules and composed of five subunits.

  • There are 10 possible alpha and 4 beta subunits
  • Nm = alpha 1 and beta 1
  • Nn = alpha 2 - 10 and beta 2 - 4

  
• Neuromuscular junction (NMJ): a specialised form of synaptic transmission - communication between neurons and skeletal muscle.
• NMJ functions:

  • When action potential arrives, voltage-gated ion channels open (by depolarisation)
  • Calcium uptake triggers neurotransmitter release which then bind with the receptor on the motor end-plate
  • More interactions with the receptor will give a stronger response
  • This forms the complex n1AChR, triggering opening of ligand-gated sodium ion channels giving muscle contraction

  
• Diseases associated with NMJ disorders:

  • Myasthenia gravis
  • Lamber-Eaton myasthenic syndrome
  • Neuromyotonia (Isaac's syndrome)
• The calcium channel is important for release of vesicles - no calcium influx means no release of neurotransmitters and no muscle relaxation/contraction.
• Blocking activity of AChE reduces the effect of ACh, and too much ACh causes no relaxation of muscle.
• Voltage-gated potassium channels (VGKCs): transmembrane channels specific for potassium and sensitive to voltage changes. They return the depolarised cell to a resting state.
• Myasthenia gravis: known as "grave muscle weakness".

  • Binding of Ach is blocked by autoantibodies and muscle activation is inhibited
  • Autoantibodies induce complement-mediated degradation of AChRs, resulting in progressive weakening of skeletal muscle
  • Antibodies move to MuSK
  • Symptoms include drooping of the face, problems with chewing, talking and swallowing
  • Progressive disease as muscles that control breathing and neck and limb movements can be affected

  
• Muscle specific tyrosine kinase (MuSK): a receptor tyrosine kinase required for formation and maintenance of the NMJ.

  • Activated by agrin (ligand)
  • Signals via proteins CK2, Dok-7 and rapsyn to induce clustering of AChR
• Lamber-Eaton myasthenic syndrome:

  • Autoantibodies to presynaptic voltage-gated calcium channel (VGCC)
  • Interferes with calcium dependent release of ACh from the presynaptic membrane causing a reduced endplate potential on the postsynaptic membrane
  • Results in NMJ transmission failure
• Neuromyotonia (Isaac's syndrome): autoantibodies to presynaptic VGKC. This is caused by antibodies binding to potassium channels resulting in continuous/hyperexcitability.
• Rocuronium: non-depolarising blocking agent, competitive inhibitor. A safer alternative to tubocurarine that gives flaccid, relaxed paralysis. It can be reversed by AChE inhibitors and gives non-NMJ effects at high dose (muscarinic blocking, histamine release).
• Succinylcholine/suxamethonium: occupies both receptor and blocks channel (ACh). Normal closure of channel is blocked, can cause desensitisation.

  • Phase 1 block: membrane depolarisation and transient fasciculations followed by paralysis
  • Phase 2 block: desensitisation, membrane repolarises and is hyposensitive to ACh
  • Cannot be reversed by AChE inhibitors

  
• Toxins such as cobratoxin or a-bungarotoxin bind with high affinity to nAChR. Drugs such as atropine can be used to reverse the reaction.
• Paralysis will begin in small, rapidly moving muscles (eyes, fingers), then limbs, last is respiratory muscles. Recovery is in reverse order.
• Tubocurarine/dimethyltubocurarine (metocurarine): blocks at ACh receptor, no effect on nerve transmission but muscle can still be stimulated.

  • 5 - 10 mg induces flaccid paralysis
  • 10 - 20 mg can produce apnoea, not active orally
  • Can cause histamine release and block ganglionic receptors at high concentration
• Alcuronium: more potent than tubocurarine, but reduced histamine release than curare and lack of ganglionic blockade.
• Gallamine: non-depolarising muscle relaxant, blocking AChRs. Can give some muscarinic block.
• Mivacurium: non-depolarising NMJ blocking drug. Used to facilitate intubation and relax skeletal muscle during surgery. Short acting, can be hydrolysed by AChE.
• Atrcurium: similar mechanism and use to mivacurium. Can be hydrolysed by AChE. Effects are greatest at about 4 minutes and last up to an hour - administered by IV.
• AChE inhibitors:

  • Increases availability of ACh to partially overcome decreased receptor availability
  • Reverses non-depolarising muscle blockade
  • First line for ocular myasthenia
  • Edrophonium, neostigmine, pyridostigmine, distigmine (in order of increasing duration of action)
  • Adjunct to immunosuppression for generalised myasthenia
  • Organophosphates make irreversible bonds to AChE

  
• Cholinergic crisis: caused by excess ACh, nerve gas, disease or surgery. Muscles stop responding to excess ACh.

  • Vasodilation of blood vessels
  • Sweating
  • Effect on muscarinic receptors
• Nerve gas poisoning: causes overstimulation of muscles, organs and glands. Symptoms include ataxia, slurred speech, areflexia, convulsions, respiratory failure and death. Treated with atropine (binds to AChR) and oximes (to regenerate AChE).
• Organophosphates are irreversible AChE inhibitors that bind covalently by phosphorous to a serine hydroxyl group in the active site to inactive AChE.
• Neostigmine: AChE inhibitor that prevents ACh breakdown. Used to treat myasthenia gravis and anaesthesia. Administration via IV into vein, muscle or skin.
• Edrophonium: reversible AChE inhibitor. Is used in the Tensilon test and is a competitive inhibitor.
• Tensilon test: a test to differentiate between myasthenic crisis and cholinergic crisis. Either ACh or edrophonium is used.

  • Improvement of symptoms by accumulation of ACh confirms myasthenia gravis
  • Worsening of symptoms shows cholinergic crisis (atropine must be administered immediately)
• Physostigmine/eserine: a reversible pseudo-competitive AChE inhibitor. Used to treat glaucoma and delayed gastric emptying. Can cross the BBB so sued to treat effects of atropine and other anticholinergic drug overdoses.
• AChE in dementia:

  • Cognitive decline is linked to loss of cholinergic transmission
  • Loss of choline acetyl-transferase and loss of cholinergic (presynaptic) neurons in forebrain
  • Muscarinergic agents are ineffective and poorly tolerated
  • Nicotinergic agonists have vascular side effects
• Tacrine: centrally active non-competitive reversible AChE inhibitor. 20 - 30% improvement in dementia.
• Donepezil: piperidine based reversible AChE inhibitor with a long half life. Used for dementia treatment.
• Rivastigmine and neostigmine: carbamate AChE inhibitors. Inactivates enzyme for 10 hr producing pseudo-irreversible inhibition. Used for dementia treatment.
• Galantamine: phenanthrene alkaloid used for dementia treatment. Competitive, reversible AChE inhibitor that also modulates nicotinic receptors.
• Antispasmodics: include botox or botulinum toxins - breakdown the SNARE complex. This blocks vesicles from releasing ACh.
• Botulinum toxin: used to treat a number of disorders characterised by overactive muscle movement, such as cerebral palsy, spasms, as well as relaxing of clenched muscles.
• SNARE protein: protein family that mediate the fusion of vesicles with the target membrane, mediating exocytosis.