Pursuing
the Holy Grail of Anesthesia [back]
The History of the University of Pittsburgh
Department of Anesthesia’s Research Program
into the Mechanism of Anesthetic Action
By Ernesto A. Pretto, Jr., MD, MPH
 Since
the discovery and introduction of ether into surgical practice by
William Morton in Boston in 1846 medical scientists have puzzled
over the exact mechanism of anesthetics.
The
history of this discovery is itself a fascinating story and I
refer those interested in learning more about it to the excellent
book entitled ‘Ether Day’ by Julie M. Fenster. (1)
Without
question anesthesia made possible the development of modern surgery
and is one of the greatest American contributions to medicine.
But despite its discovery in the United States, anesthesiology
first emerged as a medical specialty in England, because of Chloroform,
and through the efforts of the great epidemiologist and physician
John Snow (2).
Chloroform, introduced in England in the practice of Obstetrics
and known as the ‘Queen’s anesthetic’ was less
safe to use than ether, being associated with a much higher incidence
of cardiopulmonary arrest. Because of this fact only physicians
were entrusted with the task of administering it during surgery.
In the United States, however, where ether was the dominant anesthetic
surgeons delegated its administration to nurses and, as a result,
the nursing practice of anesthesia was born. (3)
This year approximately 20 million patients in the United States
alone will undergo anesthesia.
Many advances in the field have been made in the last 157 years
(4),
and yet, one of the greatest discoveries of mankind, is still
an inescapable embarrassment for the scientific community because
its action is unknown. In the past limitations were an incomplete
understanding of neuronal function and the lack of molecular tools
to investigate events at the cellular and sub-cellular level,
where anesthetic action occurs. In the past 10 years, however,
great strides have been made in our understanding of cell biology
and we are now entering a new era in this research, and are beginning
to identify the primary molecular mechanisms of anesthetic action.
The purpose of this article is not to provide a comprehensive
and detailed review of the field but rather a general overview
of the subject matter, with a focus on our own department’s
contributions over the years. I will briefly present the leading
theories of anesthetic action, mention a few of the most important
and promising molecular targets currently under investigation,
and briefly present the history of our departments efforts in
this area of research.
Hypothetical Framework
The anesthetic state is multidimensional, consisting of unconsciousness,
amnesia, analgesia, and characterized by a suspension of sensory
neural processing and depression of spinal cord reflexes. These
actions are concentrated principally in the central nervous system
(CNS). One of the fundamental questions in this field has been
whether there is a uniform theory to explain all anesthetic action.
In other words, do anesthetics exert their effects uniformly throughout
the CNS or are distinct areas of the brain affected through different
mechanisms? If so, which are directly related effects and which
are side effects? In order to explain all anesthetic effects on
the brain there have been what I will call grand unified theories
(GUT) of anesthetic action put forth. We will review several in
this article. To illustrate the scientific complexity of these
theories I have chosen one of the more provocative and intriguing
ones that have appeared recently in the literature called the
Quantum
Hypothesis of Anesthetic Action.
Lipid vs. Protein
In 1899 Meyer and in 1901 Meyer and Overton made the observation
that anesthetic potency or efficacy correlated with lipid solubility
hence the ‘Meyer-Overton correlation’:
The premise was that the accumulation of anesthetic
molecules in the neuronal cell membrane and not the chemical structure
of the anesthetic agent per se uniformly interrupted or distorted
the lipid bi-layer. In order to better comprehend this concept
visualization of the membrane is important, as follows:
In order to explain the Meyer-Overton correlation
several theories have been proposed. One is that the ‘expansion’
or thickening of the plasma membrane caused by the accumulation
or mass movement of anesthetic molecules into the membrane would
have the effect of altering gating mechanisms of ion flux into
the cell, and reversibly shutting down cell function. This idea
prevailed for 60 years and was supported by the interesting phenomenon
of reversal of anesthetic effect by increases in atmospheric pressure,
the so-called pressure reversal effect.
In the 1960s evidence began to mount about anesthetics
exerting action and interacting with membrane proteins. According
to this concept and not unlike the lipid theory proteins situated
on the surface of membranes or those present in ion channels undergo
conformational changes when exposed to anesthetics, also altering
ion flux. Therefore, anesthetic action could be characterized
as that of a membrane ‘flux capacitor’, if you will,
by inducing physicochemical changes in proteins, many of which
play an important role in cellular ion flux.
It is interesting but not surprising that research
in this area has generated more questions than it has answered.
What physicochemical changes in the cell membrane bring about
the state of general anesthesia? Is it a fluidization or a thickening
of the membrane bi-layer or both that is responsible? Is the mechanism
involved a change in the membrane lipid or the protein molecules,
or a combination of both? Although the lipid and protein theories
have not been proved or disproved conclusively current opinion
and evidence suggests that lipid solubility and/or protein perturbations
are not the sole mechanisms involved.
Molecular Mechanisms
Perhaps among the most important recent advances
in this field has been the localization of anesthetic action to
a few molecular targets in the CNS. As such, transmitter release
(6),
and ion channels or ‘molecular gates’ at the pre-,
synaptic, or post-synaptic level in dendrite and somatic cells,
and the process of axonal conductance are areas currently under
intense scrutiny. Because it is now clear that ion channels play
a key role in neuronal information processing, anesthetics must
be involved in some way in altering this process. If we wish to
fully comprehend how anesthetics work it is crucial to understand
the precise details of the gating mechanisms of ion channels.
(7)
However, a detailed discussion of this topic is beyond the scope
of this article and the interested readers are referred to the
following reviews: Molecular
Actions of General Anesthetics, as well as Ion
channels: An open and shut case.
Among the molecular mechanisms briefly mentioned
above, other more specific targets of anesthetic action that have
recently been uncovered are those affecting the neurotransmitter
glutamate and its ligand-gated ion channel, the so-called ionotropic
glutamate receptor, the G-protein receptor, also termed metabotropic
glutamate receptor – and its subclasses, and the neurotransmitter
y-aminobutyric acid (GABA). The ionotropic glutamate receptors
are divided into two classes, the AMPA/Kainate type and the NMDA
subtype. Less is known about the metabotropic receptor subtypes.
In contrast to glutamate, which causes excitation, the action
of GABA is inhibitory. Among its actions are the activation of
the GABAA and the GABAB receptors. Anesthetic
action has been observed to alter these receptors but the effects
are complicated by the fact that different anesthetics alter different
receptors and also can change ligand-receptor interaction. In
the past year two very important observations have been made concerning
these mechanisms. Evidence has been put forth that the K+ channel
is crucial for anesthetic action. (8)
In addition, an area of the brain called the tuberomammillary
nucleus or TMN in the hypothalamus has been identified as a prime
target of anesthetics. (9)
What impact these recent discoveries will have on future research
and direction of the field is not yet clear.
In summary, if we were to attempt to classify the
major foci of current research into anesthetic mechanisms we would
obtain the following research outline:
1. Molecular Basis of Anesthetic Interactions:
i. Modeling
ii. Molecular Targets
2. Anesthetic Interactions with Ion Channels and Other Proteins:
i. Nicotine Ach Receptors
ii. GABAA Receptors
iii. Other Ligand-Gated Ion Channels
iv. Potassium Channels
v. Local Anesthetics
vi. Other Proteins
3. Integration of Anesthetic Actions (In-Vitro)
And as in other branches of scientific research, work on the
molecular mechanisms of anesthetic action is shared by an international
society of scientists who, every so often, share research findings.
Preparations are afoot for the Seventh International Congress
on Molecular Mechanisms of Anesthetic Action.
University of Pittsburgh Research on Anesthetic Mechanisms
Equipped with this brief and incomplete vision of anesthetic
action I believe we can approach, at least, a fair understanding
of the significance of our department’s research in this
area. Our department’s work in this field began with Dr.
Peter Winter whose interest in the mechanism of action of anesthetics
preceded his arrival to Pittsburgh in 1979 to become the first
Safar Professor and Chairman of Anesthesia. In 1976 Dr. Winter
in collaboration with one of the undisputed giants of anesthetic
action namely Dr. Edmund ‘Ted’ Eger, and while on
sabbatical in San Francisco, demonstrated that inhalation anesthetics
are addictive drugs, in the sense that they produce, with quantifiable
predictability, both tolerance and dependence. He concluded
that this dependence was exactly analogous to that produced
by alcohol with ED50 shifts on the order of 20%, as opposed
to ED50 shifts of 200-300%, as is true for opiates. (Dr. Winter’s
Memoirs) Dr. Winter also made a brilliant observation. He demonstrated
that animals tolerant to ethanol had cross-tolerance to inhalation
anesthetics, therefore, suggesting that whatever the mechanism
of action of anesthetics, it was likely the same as that for
ethanol. Dr. Winter also performed experiments in which mice
anesthetized with barbiturates could be awakened by adding increasing
atmospheric pressure thus demonstrating the pressure reversal
effect. This was the first demonstration of pressure reversal
in animals anesthetized with an intravenous anesthetic agent.
These were groundbreaking studies and provided great insight
into the mechanisms of anesthetic action. However, the duties
of Chairman of one of the biggest anesthesia departments in
the world soon became all consuming and precluded time for more
bench research. But Dr. Winter soon recruited faculty to pick
up where he left off. One of the key individuals in the development
of the Molecular Pharmacology in Anesthesia research program
was Dr. Len Firestone, Associate Professor, and former Vice
Chairman for Research and former Department Chairman.
Since 1988 research by our department on mechanisms of anesthetic
action has been a joint collaboration between our department
and the department of pharmacology. The core group consists
of full time Ph.D. researchers Gregg E. Homanics, Yan Xu, and
Pei Tang. In addition, clinical faculty members that have worked
with the core group on various projects are Joe Quinlan and
Frank Gyulai, and in the past Leonard Firestone and Lisa Crossley.
Since becoming Chief of Anesthesia at UPMC main campus Dr. Quinlan
has devoted less time to this effort.
Among the major contributions has been a better understanding
and characterization of GABA receptor involvement in anesthetic
action. Most previous research on GABA receptors and anesthetic
action has been conducted in vitro. The introduction of genetic
engineering to the study of anesthetic mechanisms in animal
models by Gregg E. Homanics, Ph.D. facilitated the construction
of mice with mutated GABA receptors and by so doing he and others
demonstrated the altered anesthetic responses in these mice.
For example, mice lacking the b3 subunit of the GABAA receptor
display a lower sensitivity to etomidate and midazolam. This
finding provided new insight into the mechanism of action of
anesthetics at the whole animal level which is where Dr. Homanics
has concentrated his efforts. In fact Dr. Homanics states, “Because
anesthesia is defined by changes in behavioral responses, testing
of the involvement of GABA receptors in anesthetic action must
ultimately be studied at the whole animal level.” Homanics
and his team were one of the first to perform genetic dissection
of anesthetic targets by developing so-called knockout or knock-in
mice.
In general, and according to Vice Chairman for Research Dr.
Yan Xu “the field of anesthetic action has somehow been
trapped in the search for hydrophobic anesthetic binding pockets
in proteins, an idea that originated from the structure-function
paradigm (which works beautifully for enzymatic reactions and
high-affinity drug actions). But because of the diverse range
of molecules that anesthetics are and the diverse range of effects
of these molecules on proteins, the ‘unitary theory’
has been quickly (and perhaps prematurely) abandoned under the
implicit assumption that anesthetic molecules must fit into
some yet unknown structural motifs to produce protein structure
and function changes.” Drs. Pei Tang and Yan Xu, however,
believe that a common mechanism must underlie most, if not all,
molecular processes for anesthesia and adhere to the thinking
that a GUT will yet be uncovered. Their fascinating study published
recently in a paper in PNAS (http://www.pnas.org/cgi/content/full/99/25/16035)
shows unambiguously that changes in global protein dynamics
could be that common mechanism. Moreover, their results discount
the structure-function paradigm viewpoint that overrates the
importance of structural fitting between general anesthetics
and yet unidentified hydrophobic protein pockets. Dr. Yan Xu
points out, “the results underscore the global, as opposed
to the local, effects of anesthetics on protein dynamics as
the underlying mechanisms for the action of general anesthetics
and, possibly, of other low-affinity drugs.” This team
proposed the idea that susceptibility of a given protein to
anesthetic modulation (and thus the sensitivity to anesthetic)
is governed by protein global dynamics, an intrinsic protein
property. Dr. Xu argues, “the same anesthetic could have
different (and sometimes opposite) effects on global dynamics
on different timescales.” According to this thesis he
adds, “Only when the timescale of a given effect matches
the characteristic time of the protein function can that effect
have anesthesia consequences.”
Anesthetic mechanisms have been studied in our laboratories
using animal models but also in vivo at the level of
the intact human and primate brain. Drs. Frank Gyulai and Len
Firestone recently demonstrated in the human brain that the
volatile general anesthetic, isoflurane, modulates the conformational
state of the GABAA receptor in a dose-dependent manner.
This research used positron emission tomography (PET) and provided
the first human evidence for the GABAA-R hypothesis
of general anesthesia. According to Dr. Gyulai, studies are
in progress to test the specific hypothesis that volatile general
anesthetics affect GABAA-receptor conformation as
a function of their potency, i.e., the proportion of GABAA-receptors
driven to high affinity as measured by PET in the presence of
equipotent concentrations of desflurane (1 MAC = 6.0% atm) and
halothane (1 MAC = 0.75% atm).
Dr. Gyulai reports that nonhuman primate studies are also under
way in his laboratory to assess the relevance of anesthetic-related
enhancement of receptor function in terms of anesthetic ability
to increase inhibitory synaptic transmission. In order to prove
that a GABAA-receptor agonist-related increase in GABAA-receptor
function translates into an enhanced inhibitory transmission,
regional cerebral metabolic rate is directly measured using
18F-deoxyglucose with the use of PET. This is done
to observe whether isoflurane produces a left-shift of the muscimol-regional
cerebral metabolic rate dose-response curve.
The research work briefly described above is not only fascinating
and of a very high caliber but has helped to position the Department
of Anesthesia at the University of Pittsburgh as one of the
leading centers for mechanistic research in anesthesia. Please
join me in commending these individuals for their outstanding
work and dedication to an area of research that is challenging,
obscure, poorly understood, and as complex as any scientific
field, medical or non-medical. It is tantalizing to think that
yet-to-occur breakthroughs in this field may uncover secrets
of neuronal function that can greatly influence our understanding
of how the brain works – that these may be happening right
in our department is even more gratifying.
I would like to acknowledge and thank Drs. Peter Winter, Gregg
Homanics and Yan Xu for their help in the preparation and review
of this article.
Summary of NIH Funding for the Molecular Pharmacology
in Anesthesia Program
Large-Scale Molecular Dynamics Simulations of General
Anesthetic Effects on Ion Channel in Fully Hydrated Membrane:
Implication of Molecular Mechanisms of General Anesthesia
Pei Tang, PhD
Yan Xu, PhD
NIH to Dr. Tang (R01GM56257) and to Dr. Xu (R01GM49202) and
from the NSF through Pittsburgh Supercomputing Center to Dr.
Tang (MCB990034P).
NMR Structures of Second Transmembrane Domain of
Human Glycine Receptor a1 Subunit: Model of Pore Architecture
and Channel Gating
Pei Tang, PhD
Pravat Mandal, PhD
Yan Xu, PhD
Funded by NIH grants to Dr. Tang (R01GM56257) and to Dr.
Xu (R01GM49202).
Effects of Volatile Anesthetic on Channel Structure
of Gramicidin A
Pei Tang, PhD
Pravat Mandal, PhD
Martha Zegarra
Funded by a grant from the NIH to Dr. Tang (R01GM56257).
Systemic Gene Delivery in Adult Mouse Brain by Adeno-Associated
Viral Vectors
Lei Wang, PhD
Yan Xu, PhD
Funded by a grant from the NIH to Dr. Xu (R01NS36124).
Gregg Homanics:
Ethanol Mechanisms in GABAA-R Gene Targeted Mice
RO1 AA10422
NIH/NIAAA
Genetic dissection of anesthetic mechanisms
PO1 GM47818 (Eger)
Subproject (Homanics)
NIH/NIGMS
GABAa-R alpha4 subunit in ethanol-related behaviors
RO1 AA13004-01
NIH/NIAAA
Anesthetic Mechanisms in GABAA-R Gene Targeted Mice
RO1 GM52035
NIH/NIGMS
Ferenc Gyulai:
Anesthetic Mechanisms by in vivo Brain Imaging
National Institute of General Medical Sciences (National Institutes
of Health)
May-01-00 to Apr-30-04
Anesthetic Mechanisms by in vivo Human Brain
Imaging
International Anesthesia Research Society (Clinical Scholar
Research Award)
Jan-01-00 to Dec-31-01
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