Pharmacology of Intravenous Anesthetics

General Anesthesia by Intravenous Agents

For the pedagogic purposes of this chapter, intravenous anesthetic is defined as a clinically available substance that when administered directly to the patient via the bloodstream can be used to induce or maintain a state of general anesthesia. Many injectable substances, such as antihistamines or antipsychotics (see Chapter 12 ), have obvious effects on the central nervous system (CNS) and can depress cognitive status and induce sleep. These drugs are potentially useful for several perioperative situations where sedation is required, but they are neither appropriate nor safe as primary agents for producing general anesthesia. Similarly, medications like the benzodiazepines can be classified as intravenous anesthetics yet are currently used primarily in the perioperative setting for premedication and sedation, not anesthesia. The intravenous opioids (see Chapter 17 ) form the backbone of modern surgical analgesia yet are not considered true intravenous anesthetics because awareness and recall can occur despite very high doses that produce deep sedation.

The concept of balanced anesthesia was originally used by Lundy to describe premedication and light sedation as adjuncts to regional anesthesia, but the term was almost universally adopted when nitrous oxide anesthetics supplemented with thiopental and d-tubocurarine grew in popularity in the middle of the 20th century. At present, it is usually accepted that the state of general anesthesia can best be described as a delicate balance of the following effects: unconsciousness, analgesia, amnesia, suppression of the stress response, and sufficient immobility. The relative importance of each of these separate components varies for each case depending on specific surgical and patient factors; anesthesiologists must tailor combinations of intravenous drugs to match these priorities.

Intravenous Anesthesia Mechanisms and Theory

Modern anesthetic techniques have transformed surgery from a traumatic and barbaric affair to an acceptable, routine, and essential part of modern medicine. Despite the technologic advances made in perioperative medicine and surgical techniques, the drugs administered by anesthesiologists to render patients unconscious continue to be used without a clear understanding of how they produce anesthesia. Fortunately, through recent advances in molecular pharmacology and neuroscience, clinicians and investigators understand better than ever before how anesthetic chemicals can alter the function of the nervous system.

The elucidation of general anesthetic mechanisms was not accessible to traditional pharmacology methods; anesthesia is a clinical state in which multiple behavioral endpoints are caused by a structurally diverse group of drugs. Nevertheless, it appears that certain membrane proteins ( Fig. 10.1 ) possess binding sites that interact with many of the currently used anesthetics ( Table 10.1 ). In general, the halogenated volatile anesthetic agents (see Chapter 11 ) exhibit less specificity for molecular targets than the intravenous agents.

At the cellular and network levels, intravenous anesthetics alter signaling between neurons by interacting directly with a small number of ion channels. Under normal conditions, these specialized membrane proteins are activated by chemical signals or changes in the membrane environment. Upon activation, channels modify the electrical excitability of neurons by controlling the flow of ions across the cell membrane via channels coupled with specific receptors that sense the initial signal (see Chapter 1 ).

The majority of intravenous anesthetics exert their primary clinical anesthetic action by enhancing inhibitory signaling via gamma-aminobutyric acid type A (GABA _A ) receptors. Ketamine and dexmedetomidine are notable exceptions. From a neurophysiology perspective, unconsciousness can be considered as a disruption of the precisely timed cortical integration necessary to produce what is considered the conscious state. It is interesting to note that the unconsciousness produced by ketamine is phenotypically different from that produced by the GABA-ergic agents (e.g., propofol, thiopental, or etomidate). Although many talented scientists worked diligently throughout the 20th century to discover a unifying mechanism by which diverse chemicals cause what is loosely defined as the anesthetic state, molecular investigations into the action of individual drugs have revealed that this one true “grail” does not exist. It should rather be interpreted that the anesthetized state (and its separate components) can be arrived at by any disruption of the delicately constructed and precisely timed neuronal networks that underlie the normal awake, un-anesthetized state.

GABA _A Receptors

GABA is the most abundant inhibitory neurotransmitter in the brain. GABA _A receptors represent the most abundant receptor type for this ubiquitous inhibitory signaling molecule. GABA _A receptors are broadly distributed in the CNS and regulate neuronal excitability. They appear to mediate unconsciousness, arguably the most recognizable phenotype associated with general anesthesia. There is also strong evidence that GABA _A receptors are involved in mediating some of the other classic components of general anesthesia, including depression of spinal reflexes and amnesia. The contribution of GABA _A receptors in mediating immobility and analgesia is less clear.

GABA _A receptors are ligand-gated ion channels, more specifically members of the “Cys-loop” superfamily that also includes nicotinic acetylcholine, glycine, and serotonin type 3 (5-hydroxytryptamine type 3) receptors. Each of these receptors is formed as a pentameric combination of transmembrane protein subunits (see Fig. 10.1 ). This superfamily is named for the fixed loops formed in each of the subunits by a disulfide bond between two cysteine residues. In 2014, the protein crystallization of a human GABA _A receptor was reported. Binding pockets for neurotransmitters are located at two or more extracellular interfaces: in the case of GABA _A receptors, the endogenous ligand GABA binds between the α and β subunits. Thus far, 19 genes have been identified for GABA _A receptor subunits (α1-6, β1-3, γ1-3, δ, ε, θ, π, ρ1- 3). Although millions of subunit arrangements are possible, only a subset of receptor configurations is expressed in significant amounts in the CNS. Preferred subunit combinations distribute among different brain regions and even among different subcellular domains. Each type of GABA _A receptor exhibits subtly distinct biophysical and pharmacologic properties that in turn have diverse influences on synaptic transmission and synaptic integration.

Specific behavioral effects of drugs have been linked to different subunit assemblies present in different brain regions. For example, benzodiazepines are thought to interact with GABA _A receptors between the α and γ subunits (see Fig. 10.1 ) and specific clinical effects have been linked to receptors containing specific α and β subunits. Propofol, etomidate, and barbiturates interact with GABA _A receptors within, or proximal to, β subunits. The β subunits show less specific subcellular localization compared to α subunits, and the distribution of β subunits in mammalian brain does not share the same clear distinctions as α subunits. Therefore distinguishing specific differences between the GABA-mediated anesthetic effects of non-benzodiazepine intravenous anesthetics has been more elusive. However, research involving genetically manipulated mice has suggested that the sedation produced by etomidate can be primarily associated with activity at the β ₂ subunit while unconsciousness produced by the same drug can be associated with β ₃ subunits (see “ GABA _A Insights from Mutagenic Studies ”). Subtle pharmacodynamic differences between intravenous anesthetics (e.g., effects on postoperative nausea and vomiting) might also be mediated by interactions with targets in addition to GABA _A receptors.

Almost all general anesthetics enhance GABA _A receptor–induced chloride currents by enhancing receptor sensitivity to GABA, thereby inhibiting neuronal activation. Most of these drugs, at high concentrations, also directly open the channel as an agonist in the absence of GABA. By virtue of the specific distribution of these receptors in the cerebral cortex and other brain regions, in addition to being involved in producing anesthesia, GABA _A receptors function in thalamic circuits necessary for sensory processing and attention, hippocampal networks involved in memory, and thalamocortical circuits underlying conscious awareness. Computational neuronal modeling studies have been important in revealing the impact of propofol and etomidate on dynamic changes in these networks.

GABA _A Insights From Mutagenic Studies

The critical role of the GABA _A receptor in the pharmacodynamics of anesthetic drugs has been established through laboratory experimentation using genetic modifications of this protein. In the early 1990s, in vitro studies aimed at determining the interactions of the endogenous ligand GABA with its receptor revealed that specific amino acid substitutions in the GABA _A receptor conferred the sensitivity of the channel to agonist or allosteric modulator activity. By 1995, several functional domains of the GABA _A receptor associated with binding of agonists and allosteric modulators were identified (reviewed by Smith and Olsen ). Following the discovery of the site of benzodiazepine action, investigators discovered that mutation of a pair of transmembrane amino acids on the α subunit of GABA _A renders the receptors insensitive to inhaled anesthetics. That same year, a corresponding area on the β ₃ subunit was determined to be critical for the action of etomidate. Further studies have determined this location to be essential to the actions of other intravenous agents such as propofol and pentobarbital. A different amino acid residue appears to be involved with the specific actions of propofol only ( Fig. 10.2 ).

The strategy behind these discoveries was to examine the subtle differences among the amino acid sequences in receptor isoforms known to have different sensitivities to anesthetics. Originally receptor chimeras of unnatural subunit configurations were used, eventually giving way to specifically modified subunits via site-directed mutagenesis. Potential residues and amino acid sequence domains that mediate anesthetic sensitivity can be anticipated by comparing data among different mutated and chimeric receptors. Typically, functional studies are carried out in vitro by virally transfecting this DNA into living cells, which results in expression of these receptors on their cell surface. Immortal cells derived from human embryonic kidney cells and the eggs of the amphibian Xenopus laevis (oocytes) have been invaluable as conduits for this type of research; establishing electrical access in these cells using the conventional patch-clamp technique is relatively straightforward and measurements of their chloride currents in response to opening of the GABA _A channel is robust (see Fig. 10.2 ).

By now a great body of literature exists in which anesthetic sensitivity has been mapped to specific point mutations on GABA _A receptors. Some of these point mutations, so-called silent mutations, do not affect the natural function of the receptor but only its modulation by specific anesthetics. The introduction of mutations such as these into genetically modified mice can be used to determine the importance of this receptor to the production of certain qualities of an anesthetic in vivo. In vivo experimentation on animals possessing these “knock-in” point mutations has advantages over traditional gene knockout studies. Specifically, if the receptor is unaltered except with respect to its response to exogenous anesthesia, there exists less potential for compensatory changes that could influence the results of in vivo experiments. Mouse geneticists have successfully bred mice with attenuated sensitivity to benzodiazepines as well as etomidate and propofol.

By creating a point mutation that confers anesthetic selectivity in a single specific subunit, the phenotypic effects attributable to that drug and that subunit can be dissected ( Fig. 10.3 ). For example, the substitution of a methionine (M) residue for an asparagine (N) residue in the 265th position on the β ₃ subunit (β ₃ N265M mutation) results in a phenotypically normal animal that is essentially immune to the hypnotic effects of propofol and etomidate while maintaining its sensitivity to inhaled anesthetics. Similarly, substituting an arginine (R) for the histidine (H) residue in the 101st position on the α ₁ subunit (α ₁ H101R mutation) essentially renders that GABA _A receptor unresponsive to benzodiazepines. By making analogous substitutions in the other α subunits, scientists have been able to map particular behavioral effects from benzodiazepines to specific subunits. For example, the α ₁ subunit appears to mediate the sedative, amnestic, and anticonvulsant actions of some benzodiazepines, whereas benzodiazepine muscle relaxation and anxiolysis are mainly mediated via α ₂ and α ₃ subunits.

These amino acid substitutions (in vitro and in vivo) alter the molecular environment in the anesthetic binding cavity by reducing the number of favorable interactions between the receptor and the anesthetic molecule, thus reducing the efficacy of that anesthetic drug on enhancing GABA-ergic transmission. Although this work has greatly increased insight into anesthetic mechanisms, a complete description of the biophysical interactions between specific residues and specific anesthetics remains as incomplete as understanding of the spike-coded pattern of neuronal network activity responsible for the transitions between consciousness and unconsciousness.

N-Methyl-d-Aspartate Receptors

Whereas augmentation of endogenous inhibitory chemical signaling is important to mechanisms of anesthesia, mitigation of excitatory signaling also depresses neuronal activity. Of the many excitatory chemical signals in the CNS, blockade of the N-methyl-D-aspartate (NMDA)-type glutamate receptor appears to be most relevant to mechanisms of anesthesia. There are two broad categories of excitatory synaptic receptors that use the amino acid L-glutamate as their chemical messenger: NMDA receptors and non-NMDA receptors. The latter group, which mediates fast excitatory postsynaptic currents, can be subdivided into α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors and kainate receptors. Neither AMPA nor kainite receptors have a clear effect on anesthetic-induced unconsciousness, but the role of AMPA receptors in “off-target” effects of anesthetics is being actively explored (see “ Emerging Developments ”). NMDA receptors, by contrast, mediate excitatory postsynaptic currents of relatively prolonged duration. NMDA receptors are found presynaptically, postsynaptically, and extrasynaptically ; they are important targets for xenon, nitrous oxide, and the dissociative anesthetic ketamine.

NMDA receptors are tetramers consisting of four subunits arranged circumferentially around a central ion channel pore (see Fig. 10.1 ). All NMDA receptors contain an obligatory NR1 subunit and at least one of four types of NR2 subunits (A–D). Other subunits (NR3) and numerous splice variants exist that translate to considerable variability in the kinetic and pharmacologic profile of each receptor isoform. In contrast to non-NMDA glutamate receptors, which are selective for sodium ions (Na ⁺ ), the pore of NMDA receptors permits entry of both monovalent and divalent cations (Na ⁺ and calcium ions [Ca ²⁺ ]) into the cell upon activation. The Ca ²⁺ flux is important for activating Ca ²⁺ -dependent processes in the postsynaptic cell such as long-term potentiation, a form of synaptic plasticity thought to play an important role in memory.

NMDA receptors are unique among ligand-gated ion channels in that their probability of opening depends not only on presynaptic release of neurotransmitter but also on the voltage across the membrane containing the receptor. Until membrane depolarization occurs, magnesium ions (Mg ²⁺ ) block the channel pore if agonist is present. High-frequency excitatory input causes membrane depolarization, so NMDA receptors play a significant role in CNS functions that require activity-dependent changes in cellular physiology such as learning and processing of sensory information. In the nociceptive circuitry of the spinal cord, repeated peripheral nerve stimulation results in an increase in response to subsequent stimuli through activation of NMDA receptors. This “windup” phenomenon is associated with hyperalgesia, and both knockout and knockdown of NR1 block inflammatory pain in animals. This, combined with their ability to modify opioid tolerance, makes NMDA receptor antagonists promising treatments for chronic pain.

NMDA antagonists are classified by their mechanism of action. Volatile anesthetics may exert some of their effects as competitive antagonists by displacing the coagonist glycine from NMDA receptors. The intravenous NMDA antagonists in current clinical use are primarily channel blockers that bind to the pore only in its open confirmation. They are considered uncompetitive antagonists and, because their binding requires prior activation by agonist, they are termed use-dependent . Blockers such as ketamine, which remain bound after channel closure, cause prolonged disruption of the associative aspects of neuronal communication. High concentrations of ketamine cause sedation and loss of consciousness with a significant incidence of dysphoric effects. This may reflect a lack of selectivity among various NMDA isoforms or activity at other receptors (see Table 10.1 ). Noncompetitive antagonists, which bind to allosteric sites with some NMDA receptor subunit specificity, may have more specific clinical effects and a better side effect profile.

Other Molecular Targets

Other receptors, such as glycine receptors, voltage-gated Na ⁺ channels, and two-pore domain potassium (2PK) channels, deserve attention as they probably contribute to certain components of the balanced anesthetic state with intravenous anesthetics. Glycine receptors colocalize with GABA _A receptors near the cell body. Propofol, etomidate, and thiopental all have some positive modulation of the glycine receptors, but ketamine does not. Glycine receptors have an inhibitory role, particularly in the lower brainstem and spinal cord. They are likely major contributors to anesthetic immobility, especially that produced by the volatiles. An investigation of spinal neurons estimated that propofol's effects on immobility were mediated almost entirely via GABA receptors, whereas the immobility caused by sevoflurane was predominantly mediated by glycine receptors. Propofol inhibits some subtypes of voltage-gated Na ⁺ channels, which could contribute to its antiepileptic activity. In high doses, ketamine can also block Na ⁺ channel activity.

2PK Channels modulate neuronal excitability through control of the transmembrane potential. There are 15 different 2PK isoforms, and functional channels are formed from homomeric or heteromeric dimers. Genetic deletion of several members of this channel family (TREK1, TREK2, TASK1, TASK3, and TRESK) in animal models reduces the immobilizing effect of intravenous and volatile general anesthetics, which suggests a contribution to their anesthetic mechanisms.

Hyperpolarization-activated cation channels (HCN channels) are important in mediating coordinated neuronal firing between the thalamus and cortex. These channels play an important role in setting the frequency of thalamocortical rhythms critical for high-order cognitive processing and are also important for controlling burst firing in the hippocampus, thalamus, and locus coeruleus. Amnesia and hypnosis are produced upon disruption of signaling in these brain areas via inhaled and intravenous anesthetics. Some anesthetics like dexmedetomidine exert their sedating effects by activating α ₂ receptors in the locus coeruleus.

Novel techniques may identify a host of other targets for anesthetic drugs. For example, azipropofol is an analog that becomes covalently linked to a binding partner when exposed to light. Tadpoles given this drug had prolonged anesthesia when exposed to light and proteomic analysis of the linked molecules identified novel potential targets of propofol.