1. General Anesthesia: A more complex mechanism The Meyer-Overton correlation and new research into the mechanism of action of general anesthesia.

2. Purposes of General Anesthesia: (Inhaled and Intravenous) Amnesia Amnesia Analgesia Analgesia Immobility (muscle relaxation) Immobility (muscle relaxation) Loss of consciousness Loss of consciousness Hypnosis Hypnosis Suppression of noxious reflexes Suppression of noxious reflexes

3. Pharmacological Manipulation of the Neuronal Nexus Various areas of CNS mediate desired effects Various areas of CNS mediate desired effects Unconsciousness Unconsciousness Common mechanism with aspects of consciousness Common mechanism with aspects of consciousness Cerebral cortex, thalamus, and reticular formation Cerebral cortex, thalamus, and reticular formation High density of γ-aminobutyric acid (GABA-A), N-methyl-D-aspartate (NMDA) and acetylcholine (Ach) receptors High density of γ-aminobutyric acid (GABA-A), N-methyl-D-aspartate (NMDA) and acetylcholine (Ach) receptors Subject to input from subcortical arousal systems Subject to input from subcortical arousal systems

4. Amnesia Amnesia Hippocampus, amygdala and prefrontal cortex Hippocampus, amygdala and prefrontal cortex Implicit memory: recalled unconsciously (target of anesthesia) Implicit memory: recalled unconsciously (target of anesthesia) Explicit memory: recalled consciously Explicit memory: recalled consciously Use NMDA and non-NMDA receptors Use NMDA and non-NMDA receptors Respond to NT glutamate and serotonergic interneurons Respond to NT glutamate and serotonergic interneurons

5. Immobility Immobility Sensory and motor neurons Sensory and motor neurons GABA-A receptor GABA-A receptor Glutamate receptors for NMDA, alpha- amino-5-methyl-3-hydroxy-4-isoxazole propionic acid (AMPA) and kainite Glutamate receptors for NMDA, alpha- amino-5-methyl-3-hydroxy-4-isoxazole propionic acid (AMPA) and kainite Analgesia Analgesia Nocioception Nocioception Blocking occurs at glutamate, GABA-A or (micro) receptors in spinal cord Blocking occurs at glutamate, GABA-A or (micro) receptors in spinal cord

6. Meyer-Overton Correlation Has been used to describe the mechanism of volatile anesthetics Has been used to describe the mechanism of volatile anesthetics Linear relationship between potency and lipid solubility Linear relationship between potency and lipid solubility No longer accepted universally No longer accepted universally Does appear in different levels of CNS integration Does appear in different levels of CNS integration Molecular, subcellular and cellular mainly Molecular, subcellular and cellular mainly

7. Current Views of Anesthetic Mechanism Solubilization within the neuronal membrane Solubilization within the neuronal membrane Redistribution of lateral pressures Redistribution of lateral pressures Alters conformation of membrane proteins (i.e. Na + pump) Alters conformation of membrane proteins (i.e. Na + pump) Anesthetics interact with many hydrophobic sites Anesthetics interact with many hydrophobic sites Protein structures that form ion channels Protein structures that form ion channels

8. Inhaled anesthetics act at lipid bilayer-protein interface Inhaled anesthetics act at lipid bilayer-protein interface Weak electrostatic forces between membrane protein and anesthetic Weak electrostatic forces between membrane protein and anesthetic Stimulation of K + leak channels (neuronal hyperpolarization) Stimulation of K + leak channels (neuronal hyperpolarization) Ca +2 sensitivity to general anesthesia Ca +2 sensitivity to general anesthesia

9. Presynaptic Inhibition Three mechanisms of presynaptic inhibition Three mechanisms of presynaptic inhibition Mediating neuron causes Ca +2 channels of presynaptic neuron to close (< release of NT) Mediating neuron causes Ca +2 channels of presynaptic neuron to close (< release of NT) Ligand-gated receptors inhibit NT release Ligand-gated receptors inhibit NT release Ca +2 independent (botulinum/tetanus) Ca +2 independent (botulinum/tetanus) Activate GABA-A gated Cl - channels Activate GABA-A gated Cl - channels Also evidence that background K + current (upon anesthetic induction) hyperpolarizes both pre/postsynaptic neurons Also evidence that background K + current (upon anesthetic induction) hyperpolarizes both pre/postsynaptic neurons

10. Postsynaptic Inhibition Mediating neuron hyperpolarizes another neuron Mediating neuron hyperpolarizes another neuron Agonist binds to postsynaptic GABA-A receptor Agonist binds to postsynaptic GABA-A receptor

11. Inhibitory Pathways GABA GABA Key inhibitory NT within the brain Key inhibitory NT within the brain Two types (A and B) Two types (A and B) GABA-A receptors increase Cl - conductance (postsynaptic) GABA-A receptors increase Cl - conductance (postsynaptic) Analogous ligands (agonists) aside from GABA interact with GABA receptors Analogous ligands (agonists) aside from GABA interact with GABA receptors Benzodiazepines, barbiturates, anesthetic steriods, volatile anesthetics and ethanol Benzodiazepines, barbiturates, anesthetic steriods, volatile anesthetics and ethanol

12. GABA-A/B/C GABA-A: individual expression of the GABA-A receptor subunit composition and subunit isoforms can modify response to anesthetic GABA-A: individual expression of the GABA-A receptor subunit composition and subunit isoforms can modify response to anesthetic GABA-B: linked via G proteins to K + channels GABA-B: linked via G proteins to K + channels Activated—GABA-B receptors decrease Ca +2 conductance and inhibit cAMP production Activated—GABA-B receptors decrease Ca +2 conductance and inhibit cAMP production No KNOWN association with anesthesia No KNOWN association with anesthesia GABA-C: also ligand-gated Cl - channels GABA-C: also ligand-gated Cl - channels

13. GABA-A Receptor GABA-A receptors contain various subunits within the predominate structure GABA-A receptors contain various subunits within the predominate structure 1-6 α, 1-4 β, 1-4 γ, δ, ε, 1-2 ρ 1-6 α, 1-4 β, 1-4 γ, δ, ε, 1-2 ρ 70-70 kDa glycoprotein 70-70 kDa glycoprotein Contains 12 hydrophobic membrane-spanning domains Contains 12 hydrophobic membrane-spanning domains Two other GABA receptors (B and C) Two other GABA receptors (B and C)

14. GABA-A Receptor GABA Cl - Axoplasm Voet and Voet 2 nd Edition

15. GABA-A Inhibition Increase in Cl ion conductance after activation of GABA-A receptors by anesthesia Increase in Cl ion conductance after activation of GABA-A receptors by anesthesia Causes localized hyperpolarization of the neuronal membrane Causes localized hyperpolarization of the neuronal membrane Increased threshold to depolarize (to form AP) Increased threshold to depolarize (to form AP) Increased conductance is due to an increase in the mean open time of the Cl ion channel Increased conductance is due to an increase in the mean open time of the Cl ion channel

16. Formation of GABA Initial step utilizes α-ketoglutarate (Krebs) Initial step utilizes α-ketoglutarate (Krebs) Transamination of α-ketoglutarate to form α-oxoglutarate transaminase (GABA-T or glutamate) Transamination of α-ketoglutarate to form α-oxoglutarate transaminase (GABA-T or glutamate) Glutamate is decarboxylated to form GABA by glutamate decarboxylase (GAD) Glutamate is decarboxylated to form GABA by glutamate decarboxylase (GAD)

17. Degradation of GABA Metabolized by GABA-T to form succinic semialdehyde Metabolized by GABA-T to form succinic semialdehyde Glutamate is regenerated if in the presence of α- ketoglutarate Glutamate is regenerated if in the presence of α- ketoglutarate If not, succinic semialdehyde is oxidized by SSADH then succinic acid returns to Krebs cycle If not, succinic semialdehyde is oxidized by SSADH then succinic acid returns to Krebs cycle

18. Off Topic Off Topic GAD is also present in β cells of pancreatic islets GAD is also present in β cells of pancreatic islets GAD plays role in pancreatic endocrine function GAD plays role in pancreatic endocrine function Insulin and GAD coexist in the β cells Insulin and GAD coexist in the β cells Antibodies of the 64-kDa (GAD) occur in almost all patients with insulin-dependent diabetes Antibodies of the 64-kDa (GAD) occur in almost all patients with insulin-dependent diabetes Presence of GAD antibodies appear to precede the clinical onset of the disease Presence of GAD antibodies appear to precede the clinical onset of the disease GAD and development of Type-1 diabetes??? GAD and development of Type-1 diabetes???

19. Glycine Receptor Ogliomeric transmembrane protein composed of 3 α and 2 β subunits Ogliomeric transmembrane protein composed of 3 α and 2 β subunits Agonists: β-alanine and taurine as well as β- aminobutyric acid, ethanol and anesthetics as well as strychnine Agonists: β-alanine and taurine as well as β- aminobutyric acid, ethanol and anesthetics as well as strychnine Isofluorane and propofol are also allosteric effectors Isofluorane and propofol are also allosteric effectors Similar in structure to GABA-A receptor Similar in structure to GABA-A receptor GLYT-1 and GLYT-2 GLYT-1 and GLYT-2

20. Receptor consists of two polypeptide subunits Receptor consists of two polypeptide subunits 48 kDa (α) and 58 kD (β) 48 kDa (α) and 58 kD (β) Glycine binding site is located on α Glycine binding site is located on α Each subunit has 4 hydrophobic membrane-spanning sequences Each subunit has 4 hydrophobic membrane-spanning sequences Garrett and Grisham 3 rd Edition

21. Glycine α-1 Transmembrane Domain Protein Data Bank

22. Glycine Receptor Gar Garrett and Grisham 3 rd Edition

23. K + Background (Leak) Channel Excitation Leak channels influence both resting membrane potential and repolarization Leak channels influence both resting membrane potential and repolarization These channels are opened by volatile anesthetics These channels are opened by volatile anesthetics Hyperpolarization of the membrane Hyperpolarization of the membrane Suppresses action potential generation Suppresses action potential generation Partially responsible for suppressing the hypoxic drive during general anesthesia Partially responsible for suppressing the hypoxic drive during general anesthesia

24. Hypoxic Drive Lung damage Lung damage Alveolar ventilation is inadequate Alveolar ventilation is inadequate Abnormal arterial blood gases. Abnormal arterial blood gases. Chemoreceptors become tolerant of a high pp of CO 2 ; kidneys compensate for the respiratory acidosis by retaining bicarbonate (HCO 3 ) Chemoreceptors become tolerant of a high pp of CO 2 ; kidneys compensate for the respiratory acidosis by retaining bicarbonate (HCO 3 ) Keeps arterial pH normal Keeps arterial pH normal If Too much oxygen respiratory drive will be lost If Too much oxygen respiratory drive will be lost Not breathe adequately, Not breathe adequately, Pp of CO 2 in arterial blood will rise (loss of consciousness) Pp of CO 2 in arterial blood will rise (loss of consciousness)

25. Disruption of Ligand Diffusion Chreodes A proposed mechanism of action for inhaled anesthetics

26. Diffusion Chreodes What the %$#* are Chreodes you ask? Protein cavities are targeted by anesthetic molecules Protein cavities are targeted by anesthetic molecules This disrupts the normal function of the protein This disrupts the normal function of the protein Amino acids outside the active site act as “promoters” Amino acids outside the active site act as “promoters” These chreodes created in the landscape of the receptor are invoked to account for a type of facillitated diffusion of a ligand to that receptor These chreodes created in the landscape of the receptor are invoked to account for a type of facillitated diffusion of a ligand to that receptor Exit of ligand from active site may be mediated by another set of chreodes Exit of ligand from active site may be mediated by another set of chreodes

27. Chreodes It is believed that the viscosity of water near the protein surface is higher (due to the intermolecular forces between the amino acid side chains and the water molecules) than the “bulk” water It is believed that the viscosity of water near the protein surface is higher (due to the intermolecular forces between the amino acid side chains and the water molecules) than the “bulk” water This ordering of “layers” of water could facilitate faster diffusion of the solute (ligand) near the protein surface This ordering of “layers” of water could facilitate faster diffusion of the solute (ligand) near the protein surface These paths for the ligand are always changing until (over time) they continue to return to an ordering that promotes fastest diffusion and stability These paths for the ligand are always changing until (over time) they continue to return to an ordering that promotes fastest diffusion and stability A molecule could potentially disrupt the ordering of water and amino acid side chains disrupting the chreodes A molecule could potentially disrupt the ordering of water and amino acid side chains disrupting the chreodes

28. And Finally (I know you’re happy) Chreodes and Anesthesia Inhalational anesthetics (IA) are approximately equal in size to the AA side chains Inhalational anesthetics (IA) are approximately equal in size to the AA side chains IA have lipophilicities very close to those of lipophilic side chains IA have lipophilicities very close to those of lipophilic side chains The presence of IA in or near a chreode could alter the unique path adopted by the receptor, disrupting the normal diffusion of the ligand to the receptor The presence of IA in or near a chreode could alter the unique path adopted by the receptor, disrupting the normal diffusion of the ligand to the receptor

29. Partition Coefficients of AA Side Chains and Volatile Anesthetic Drugs Tryptophan 2.25; Sevoflurane 2.34 Tryptophan 2.25; Sevoflurane 2.34 Isoleucine 1.80; Phenylalanine 1.8; Desflurane 1.80 Isoleucine 1.80; Phenylalanine 1.8; Desflurane 1.80 Leucine 1.70; Halothane 1.70 Leucine 1.70; Halothane 1.70 Tyrosine 0.96; Ether 0.89 Tyrosine 0.96; Ether 0.89