Presentation by: Kristen Stout

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Effects of the antibiotic gentamicin on nicotinic
acetylcholine receptorsMascia Amici, Fabrizio
Eusebi, Ricardo Miledi

• Presentation by Kristen Stout

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Aminoglycosides

• Gentamicin, kanamycin, streptomycin, etc.
• Typically used to treat gram-negative bacterial
  infections.
• Known to cause ototoxicity, nephrotoxicity, and
  neuromuscular block (Chambers and Sande, 1996)

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Gentamicin

• Commonly used aminoglycoside
• Believed to target nAChRs and
  thus cause observed side
  effects.
• Previous research has shown that gentamicin
• Acts as a competitive antagonist/open channel
  blocker of recombinant muscle AChRs.
• Accelerates desensitization of muscle AChRs.
• May cause reversible paralysis through a combined
  action on pre- (inhibiting ACh release) and
  post-synaptic (blocking AChRs) AChRs.

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Questions

• Does prolonged treatment with gentamicin alter
  functionality of nAChRs?
• Does gentamicin act directly on AChRs or does it
  utilize a secondary messenger?
• Do observed effects of acute gentamicin
  application to muscle AChRs also apply to
  neuronal AChRs?

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Methods

• Plasmids of a, ß, ?, d, and a7 subunits were
  transformed into E. coli. Individual plasmids
  were extracted, linearized, and purified. They
  were then used as templates to synthesize capped
  RNA.
• Oocytes from X. laevis frogs were harvested and
  prepared.
• 15 nl of mRNA was injected in the following
  increments
• 1111 aß?d- rat mRNA
• All human a7 subunit

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Results

• The amplitude of ACh current was greatly reduced
  in oocytes maintained in gentamicin containing
  solution. This effect was specific and poorly
  reversible.

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Results

• The blocking action of gentamicin on IACh was
  concentration dependent.
• Gentamicin slowed the decay of Ach-current 2
  fold. This effect was fully reversible.
• Gentamicin presence causes no significant effect
  to reversal potential. The inhibitory action of
  gentamicin is voltage dependent.

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Calcium

• a7 nAChRs are highly calcium permeable.
• Voltage dependence of gentamicin-induced
  inhibition of Ach currents may be due to
  endogenous calcium activated chloride channels.
• To test this oocytes were maintained in either
  calcium free or high calcium concentration
  Barths solution.
• Results indicate that
• Chloride channels appear to contribute to the
  voltage dependence of gentamicin on a7 nAChRs.
• Acutely applied gentamicin effects are mitigated
  in the presence of high calcium concentration.

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Discussion

• Many laboratories routinely use the antibiotics
  in the work for maintaining Xenopus oocytes
  From our findings it is evident that not only
  gentamicin but also kanamycin, penicillin, and
  streptomycin alter the properties of nicotinic
  receptors. Moreover, since some of these effects
  are slowly reversible investigators should bear
  this in mind when performing studies with oocytes
  maintained in antibiotics.
• a7 inhibition due to gentamicin may be the cause
  of cochlear ototoxicity.

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Discussion

• The tests linking nicotinic receptor
  desensitization and drug side effects may be
  pharmacologically significant.
• Link between nicotinic receptor desensitization
  and muscle and vestibular dysfunction may suggest
  that such desensitization is employed in other
  pathological processes.

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References

• Chambers, H.F., Sande, M.A., 1996. Antimicrobial
  agents the aminoglycosides. In Hardman, J.G.,
  Limbird, L.E. (Eds.),Goodman Gilmans The
  pharmacological basis of therapeutics.
  McGraw-Hill, New York, pp. 1103-1121.
• Amici, Mascia Eusebi, Fabrizio Miledi,
  Ricardo., 2005. Effects of the antibiotic
  gentamicin on nicotinic acetylcholine
  receptors. Neuropharmacology. 49627-637.