Mechanisms of Action of General Anesthetics

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Mechanisms of Action of General Anesthetics

• BCP 401 - Principles of Pharmacology
• JP Dilger
• November 26, 2008

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Outline

• What is general anesthesia?
• MAC - Minimum Alveolar Concentration
• Chemical structures of general anesthetics
• Meyer-Overton correlation exceptions
• Possible anesthetic binding sites
• Molecular approaches to understanding anesthesia
• In vivo approaches to understanding anesthesia

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What is general anesthesia?
Do you think I should give him 50 or 100?
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Analgesia
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Amnesia
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Hypnosis/Sedation
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Immobility
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General Anesthesia

• Analgesia
• Amnesia
• Hypnosis/sedation
• Immobility

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How is anesthesia measured?

• Lack of purposeful response to painful stimulus
• surgical incision (humans, other mammals)
• tail clamp (rat, mouse)
• heat (rat, mouse, fruit fly)
• Loss of righting reflex (rat, mouse)
• Loss of spontaneous movement
• Swimming (goldfish, tadpole)
• flying (fruit fly)
• crawling (nematode)

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MAC Minimum Alveolar Concentration

• 1.0 MAC is the concentration of inhalational
  anesthetic required to blunt the muscular
  response to surgical skin incision of 50 of a
  population of unparalyzed patients.

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MAC the population response to anesthetics
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Halothane MAC in the Animal Kingdom
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MAC is not the whole story
MAC for isoflurane in rats Control
1.300.25 Decerebrated 1.260.14
MAC for isoflurane in goats Control
1.20.3 CNS bypass 2.90.7 Recovery 1.30.1
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Anesthetics - Halogenated ethers
MW
1
2
3
4
5
6
7
8
Diethyl ether
74
H
H
CH
H
H
H
H
H
3
Fluroxene
126
H
H
CH
H
F
F
F
2
Methoxyflurane
165
F
H
H
H
F
Cl
H
Cl
Desflurane
168
H
F
H
F
F
F
F
F
Isoflurane
184
H
F
H
F
Cl
F
F
F
Enflurane
184
F
F
H
F
F
Cl
H
F
Sevoflurane
200
H
H
F
H
CF
F
F
F
3
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Other inhalational anesthetics
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Intravenous anesthetics

• Propofol
• Etomidate
• Alphaxalone

Thiopental Midazolam Ketamine
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Anesthetic Properties
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Partition Coefficients
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Meyer Overton Correlation
HAL Halothane ISO Isoflurane ENF Enflurane SEV Sev
oflurane FLU Fluroxene DES Desflurane CYC Cyclopro
pane MOF Methoxyflurane DEE Diethylether CHL Chlor
oform DDM Dichlorodiflurormethane PFE Perfluoroeth
ane CTF Carbon tetrafluroide SHF Sulfur
hexafluoride CYC Cyclopropane ETH Ethylene NO2 Nit
rous oxide XEN Xenon NIT Nitrogen KRY Krypton ARG
Argon
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Non-anesthetics (non-immobilizers)
Correlation of 22
anesthetics in mouse
10000
1000
100
10
1
0.1
Olive Oil Gas Partition Coefficient
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Awake! Awareness during anesthesia

• 25 incidents in 19,575 cases
• 1-2 per 1000
• 26,000 per year in US

Alba Christensen, 2007
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Lipid-Based Theories of Anesthetic Action

• Direct action on lipids leads to Indirect action
  on proteins
• Thickness and/or Volume Fluidity
• Curvature Dielectric properties
• Phase Transitions Ionic Permeability

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Status of Lipid Theories

• Currently in disfavor
• little physical evidence - effects on bilayer
  properties are small
• anesthetic to lipid ratio is low
• 50 mM anesthetic distributed uniformly through
  50Å thick membrane corresponds to
• 1 anesthetic molecule per 60 lipids
• 1.5 of the molecules
• 1 of the volume
• specificity problem - lipids are ubiquitous
• what about nonimmobilizers?

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Fireflies and Anesthetics
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Protein-based theories of Anesthetic Action

• Anesthetics bind to amphipathic sites on
  proteins.
• induce/prevent conformational change
• alter kinetics of conformational changes
• compete with ligands

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Status of Protein Theories

• Currently favored by many investigators
• anesthetic binding sites have been identified on
  several proteins
• some of these sites are stereospecific
• effects on some proteins seen at relevant
  concentrations of anesthetics
• the binding of 1 anesthetic molecule to 1 protein
  could produce a significant effect if the binding
  occurred at a functionally important part of the
  protein

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Status of Protein Theories

• Problems
• lack of a chemical anesthetic antagonist
• specificity problem - proteins are ubiquitous
• which protein(s)?

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Where do anesthetics act in the CNS?
CNS
Neurons
Glia
Axons
Synapses
Channels
Receptors Pumps Second Messengers Intracellular
Enzymes ??
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Synaptic Effects of Anesthetics
Without anesthetic
With anesthetic
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Direct effects of anesthetics on ion channels

• Channel blocking effects of general anesthetics
  on nicotinic AChR.

(Dilger et al 1994)
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Nicotine Reversal of Sedation?

• Implant cannula for microinjection of nicotine
  into rat thalamus (?4?2 AChR)
• After 1 week, induce loss of righting reflex with
  sevoflurane
• Inject nicotine look for signs of arousal
• Partial eye or other purposeful movements
• Complete turning over ambulation

(Alkire et al 2007)
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Effects of Nicotine
(Alkire et al 2007)
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What about nicotine antagonists?

• They do NOT induce LORR themselves
• Consider nicotinic activity in thalamus as
  regulating an on switch but not an off switch

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An Anesthetic Binding Site on the GABAA Receptor?
Krasowski et al 1998
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Model of Binding Site
Mascia et al 2000
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Genetics

• Jurd et al 2003
• Knock-in mice b3(N265M) GABAA receptors
• Molecular biology
• Electrophysiology
• Behavioral
• Anesthetics etomidate, propofol, enflurane,
  alphaxolone

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Jurd et al. - etomidate
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Jurd et al. enflurane
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Working Hypothesis for Anesthesia
Eger et al, 1997 Solt Forman, 2007
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Milestones in Anesthetic Mechanisms

• Meyer-Overton Correlation
• Anesthetics may interact directly with proteins
• Brain vs. spinal cord
• Role of GABAA receptor for propofol and etomidate
  effects on awareness

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Summary?

• Progress in anesthesia research is severely
  hindered by
• our inadequate understanding of the brain
• the low affinity of anesthetics for their site of
  action
• the lack of a graded measure of depth of
  anesthesia
• the lack of an agent that reverses anesthesia
• many nonspecific effects

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Summary!

• Progress in anesthesia research is attainable
  because of
• the powerful tools of molecular biology,
  electrophysiology and genetics
• many people are studying many possible targets
  for anesthetics

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Bibliography

• Alkire MT, McReynolds JR, Hahn EL, Trivedi AN.
  Thalamic microinjection of nicotine reverses
  sevoflurane-induced loss of righting reflex in
  the rat. Anesthesiology 107 264-672, 2007
• Antognini JF, Schwartz K. Exaggerated anesthetic
  requirements in the preferentially anesthetized
  brain. Anesthesiology 79 1244-1249 , 1993
• Dilger JP, Vidal AM, Mody HI, Liu Y Evidence for
  direct actions of general anesthetics on an ion
  channel protein - a new look at a unified
  mechanism of action. Anesthesiology 81
  431-442, 1994
• Eger EI, Koblin DD, Harris RA, Kendig JJ,
  Pohorille A, Halsey MJ, Trudell JR Hypothesis -
  inhaled anesthetics produce immobility and
  amnesia by different mechanisms at different
  sites. Anesthesia Analgesia. 84915-918, 1997
• Franks NP, Lieb WR Molecular and cellular
  mechanisms of general anaesthesia. Nature 367
  607-614 , 1994
• Franks NP, Lieb WR Do general anaesthetics act by
  competitive binding to specific receptors? Nature
  310 599, 1984
• Jurd R, Arras M, Lambert S, Drexler B, Siegwart
  R, Crestani F, Zaugg M, Vogt KE, Ledermann B,
  Antkowiak B, Rudolph U. General anesthetic
  actions in vivo strongly attenuated by a point
  mutation in the GABAA receptor b3 subunit. FASEB
  J 17, 250, 2003
• Koblin DD, Chortkoff BS, Laster MJ, Eger EI,
  Halsey MJ, Ionescu P Polyhalogenated and
  perfluorinated compounds that disobey the
  Meyer-Overton hypothesis. Anesthesia and
  Analgesia 79 1043-1048, 1994
• Krasowski MD, Koltchine VV, Rick CE, Ye Q, Finn
  SE, Harrison NL. Propofol and other intravenous
  anesthetics have sites of action on the
  g-aminobutyric acid type A receptor distinct from
  that for isoflurane. Molecular Pharmacology 53
  530-538,1998
• Mascia MP, Trudell JR, Harris RA. Specific
  binding sites for alcohols and anesthetics on
  ligand-gated ion channels. Proc Natl Acad Sci U S
  A. 979305-10, 2000
• Rampil IJ, Mason P, Singh H Anesthetic potency
  (MAC) is independent of forebrain structures in
  the rat. Anesthesiology 78 707-712 , 1993
• Sebel PS, et al. The incidence of awareness
  during anesthesia A multicenter United States
  study. Anesth Analg 99833-9, 2004
• Solt K, Forman SA. Correlating the clinical
  actions and molecular mechanisms of general
  anesthetics. Curr Opin Anaesth 20300-6, 2007
• Sonner JM, Antognini JF, Dutton RC, Flood P, Gray
  AT, Harris RA, Homanics GE, Kendig J, Orser B,
  Raines DE, Rampil IJ, Trudell J, Vissel B, Eger
  EI. Inhaled anesthetics and immobility
  mechanisms, mysteries, and minimum alveolar
  anesthetic concentration. Anesth Analg.
  97718-40, 2003