Comfortably Numb

Anesthetics are slowly giving up the secrets of how they work
John Travis

Take a stroll through the Boston Public Garden, the nation's oldest botanical garden, and you'll find an array of plaques, monuments, and memorials honoring famous people of history. Not far from a statue of George Washington on horseback, there's a tall monument that honors not a person, but a chemical. This tribute to ether is probably the world's only monument to a drug. A statue representing the Good Samaritan tops the structure, which displays the inscription, "There shall be no more pain."

Erected in the 19th century, the tribute commemorates ether's first use as a surgical general anesthetic, which took place in 1846 at the Massachusetts General Hospital in Boston. Today, it's hard to imagine what surgery was like before this discovery and the subsequent development of inhaled and injected chemicals that are even more effective in rendering people unconscious and insensitive to pain.

General anesthesia has a "magical quality to it," says anesthesiologist James Sonner of the University of California, San Francisco. "It was and still is amazing that you can . . . make an organism comatose, unresponsive enough to perform surgery, and reverse the whole thing."

Almost as remarkable, scientists until recently had little solid evidence of how these drugs perform their magic. "Anesthetics have been used for 160 years, and how they work is one of the great mysteries of neuroscience," Sonner says.

Anesthesia research "has been for a long time a science of untestable hypotheses," notes Neil L. Harrison of Cornell University.

However, last year, a European research group put a leading theory to the test. The team reported using genetically engineered mice to confirm a proposed mechanism for two anesthetics that are delivered by injection. That research strategy is now being brought to bear on the more complex and contentious issue of how inhaled anesthetics work. Many investigators expect this effort to succeed, although they say that it could take 5 to 10 year.

"Most of the injectable anesthetics appear to act on a single molecular target," says Sonner. "It looks like inhaled anesthetics act on multiple molecular targets. That makes it a more difficult problem to pick apart."

Beyond the ether

Combining amnesia, sedation, immobility, and insensitivity to pain, general anesthesia is an unnatural state: a physician-induced "central nervous system dysfunction," in the words of Roderic Eckenhoff of the University of Pennsylvania in Philadelphia.

Many millions of people in the United States undergo general anesthesia annually. Seeking quicker-acting agents with fewer side effects, modern anesthetists have moved beyond ether. Among the inhaled anesthetics, ether derivatives halothane, isoflurane, and enflurane are the most common, but nitrous oxide (laughing gas), cyclopropane, and xenon, which aren't related to ether, also work. Two of the most commonly injected anesthetics are propofol and etomidate.

One of the central questions of anesthesia research has been whether all these drugs work in the same manner. Until the last few decades, investigators generally held that, despite chemical differences, the drugs share a mechanism of action.

One long-standing theory was based on the simple observation that the more soluble an anesthetic is in olive oil, the more effective it is. Drawing upon that oddity, scientists since the early 1900s argued that anesthetics suppress brain function by dissolving into and altering the structure of lipid-based membranes of nerve cells. This, in turn, would change the function of ion channels, the pores that govern the electrical activity of the cells.

In the 1970s, the lipid theory faced its first serious challenge. Researchers including Nicholas P. Franks and William R. Lieb of Imperial College in London argued that anesthetics act as traditional drugs do. That is, they bind to specific protein targets on nerve cells and activate the molecules or disrupt their function.

These scientists' initial suspicion centered on neurotransmitter receptors, the cell-surface proteins that respond to the chemical signals that nerve cells secrete. The receptors for brain chemicals such as glutamate, glycine, and gamma-amino butyric acid (GABA) drew particular attention because they control the flow of ions into nerve cells.

Glutamate is the primary excitatory signal used by these cells, whereas glycine and GABA typically shut down nerve cell activity. Therefore, anesthetics might work by blocking glutamate receptors or by activating GABA or glycine receptors. Research in the 1980s produced growing evidence that various anesthetic agents, particularly the injected ones, bind to such receptors. In tests on cells grown in the laboratory, scientists even pinpointed specific regions of the receptors where the anesthetics seemed to act.

But showing that a drug affects a protein in a laboratory dish is a far cry from proving that the drug uses that protein to produce anesthesia in a person. So, researchers turned to genetically engineered mice to address whether glutamate, glycine, or GABA receptors are the targets of anesthetics. The initial forays into this area relied on knockout mice, animals in which scientists have mutated a single gene to prevent the production of its protein.

The scientists reasoned that if a mouse lacks a particular target of an anesthetic, say a GABA receptor, it should be resistant to the drug's effects. However, most of the knockout mice lacking the receptors died as embryos, were too sick for the scientists to work with, or remained sensitive to the anesthetic being tested.

Investigators then turned to a more subtle strategy, making what they call knockin mice. In this approach, researchers change a gene's DNA sequence so that it synthesizes a slightly modified version of the protein being studied. In the case of anesthesia, for example, investigators turned to mutations that render GABA receptors insensitive to various anesthetics in studies of cells. Some of these mutations change just a single amino acid within a receptor protein, so the scientists suspect that they've altered the site on the receptor where it interacts with the drugs.

Although Harrison, then of the University of Chicago, and his colleagues suggested in 1997 that this knockin approach could confirm the molecular targets of anesthetics, it was a research team from outside the anesthesia field that first succeeded with the strategy.

Uwe Rudolph of the University of Zurich has long studied the workings of a class of antianxiety drugs called benzodiazepines, which include Valium and Xanax. These drugs appear to target GABA receptors, particularly ones called GABA-A. Three families of proteins—alpha, beta and gamma—join in various combinations to make up slightly different versions of the GABA-A receptor.

Test-tube studies had indicated that the sedative and antianxiety effects of benzodiazepines depend upon different subunits of the GABA-A receptor. In 1999, for example, the Zurich team confirmed that mice with subtle mutations in a certain alpha subunit are insensitive to the sedating properties of the drug. Other knockin mice further defined how benzodiazepines work. "Then, we set our sights on general anesthetics," says Rudolph.

He and his coinvestigators concentrated on an amino acid in a beta subunit that Harrison's group and other scientists had identified as potentially important to anesthetic sensitivity. Rudolph, his colleague Rachel Jurd, and the rest of their team created a knockin mouse bearing an amino acid–changing mutation in the beta subunit and then compared how normal mice and the knockin mice reacted to various anesthetics.

It's not easy to evaluate a mouse's response to an anesthetic, but researchers have several strategies. The simplest is to test whether a drug has blunted an animal's response to potentially pain-inducing manipulations. For example, Rudolph's group used a technique in which they pull back a mouse's hind limbs. Unanesthetized animals apparently find this painful and reflexively draw in their hind limbs.

An injection of etomidate or propofol prevented this reflex in typical mice but not in the knockin mice with the mutant GABA-A receptor, the team reported in the Feb 17, 2003 FASEB Journal. The knockin animals, however, remained sensitive to the inhaled anesthetic enflurane.

The experiment establishes that propofol and etomidate anesthetize mice by interacting with GABA-A receptors, says Rudolph.

Alex Evers of Washington University in St. Louis agrees. "It's a proof," he says. The finding is a "spectacular result" and a "triumph," says Harrison, now of Cornell University.

It's a gas

While researchers generally agree that the work of Rudolph's team, combined with the studies that led up to it, clarify how the most important injected anesthetics work, controversy still envelops the inhaled anesthetics. These compounds don't bind to molecules nearly as strongly as etomidate and propofol do. Because these anesthetics seem to alight only briefly on molecules, it's been difficult to identify the chemicals' potential targets on cells.

Receptors for glutamate and GABA are candidates because of their roles in the brain, but many other proteins have attracted attention too. In the February Molecular Pharmacology, Franks, Lieb, and their colleagues reported that xenon, nitrous oxide, and cyclopropane could, at least in test-tube experiments on cells, regulate the activity of yet another group of membrane proteins, ones that control the flow of potassium ions into cells.

While Franks and most other scientists suspect that inhaled anesthetics have, at most, a few molecular targets, Eckenhoff takes a more expansive view. Over the past few years, he and his colleagues have published a series of papers suggesting that inhaled anesthetics bind to dozens, perhaps hundreds, of proteins inside and on the surfaces of brain cells.

In the May 7 Journal of Biological Chemistry, for example, they report that halothane binds to 90 proteins, about one in every four isolated from the membranes of rat nerve cells. Such findings prompt Eckenhoff to argue that researchers seeking just a few molecular targets for inhaled anesthetics will fail.

The drugs "probably work through a constellation of small effects at lots and lots of sites," he contends.

"I don't agree," says Harrison. "There may be hundreds of proteins that the anesthetic interacts with . . . but that doesn't mean all those targets are important or relevant."

At the moment, many anesthesia investigators suspect that inhaled anesthetics such as halothane and isoflurane work through GABA-A receptors plus a few other targets. In contrast, the evidence indicates that xenon, nitrous oxide, and cyclopropane don't affect either type of GABA receptor but do interact with glutamate receptors and several other nerve cell–surface proteins.

"It's clear that anesthesia is a complex state," says Evers. "It's also clear that not all the drugs act the same way." That realization, he adds, is one of the biggest advances the anesthesia field has made.

The next steps for anesthesia researchers, says Harrison, are to create many different knockin strains of mice, each with a mutation in a different potential target molecule or subunit, and to test whether the animals are sensitive to various inhaled anesthetics. Harrison has already joined forces with Gregg Homanics of the University of Pittsburgh School of Medicine to do just that. Last fall at the annual Society for Neuroscience meeting in New Orleans, they reported that mutating the alpha subunit of the GABA-A receptor makes mice somewhat less sensitive to isoflurane and enflurane, but has no effect on the animals' susceptibility to halothane.

Since it may be that no single mutation can make a mouse completely insensitive to any inhaled anesthetic, the researchers will likely have to crossbreed the various knockins to combine target mutations in one animal that can then be tested. "It's going to take a lot of time and work," says Homanics.

While anesthesiologists are relatively happy with the drug arsenal they currently have, a clear understanding of how anesthetics work could lead to improved ones, says Sonner. The anesthesiologist also speculates that learning how the drugs put people into a reversible coma may provide insight into consciousness and other aspects of the mind.

"I wonder if we will find out something profound about the way the nervous system is designed," says Sonner.

References:

Darbandi-Tonkabon, R. . . . and A.S. Evers. 2003. Photoafffinity labeling with a neuroactive steroid analogue. Journal of Biological Chemistry 278(April 11):13196-13206. Available at http://www.jbc.org/cgi/content/full/278/15/13196.

Eckenhoff, M.F., K. Chan, and R.G. Eckenhoff. 2002. Multiple specific binding targets for inhaled anesthetics in the mammalian brain. Journal of Pharmacology and Experimental Therapeutics 300(January):172-179. Available at http://jpet.aspetjournals.org/cgi/content/full/300/1/172.

Gruss, M. . . . and N.P. Franks. 2004. Two-pore-domain K⁺ channels are a novel target for the anesthetic gases xenon, nitrous oxide, and cyclopropane. Molecular Pharmacology 65(February):443-452. Abstract available at http://molpharm.aspetjournals.org/cgi/content/abstract/65/2/443.

Harrison, N.L. 2003. Knockin' on the door of general anesthetic mechanisms: But will U.S. researchers be shut out? Anesthesia & Analgesia 97(September):616-618.

Jurd, R. . . . and U. Rudolph. 2003. General anesthetic actions in vivo strongly attenuated by a point mutation in the GABA_A receptor beta-3 subunit. FASEB Journal 17(February):250-252. Available at http://www.fasebj.org/cgi/content/full/17/2/250.

Sonner, J.M. . . . G.E. Homanics, et al. 2003. Inhaled anesthetics and immobility: Mechanisms, mysteries, and minimum alveolar anesthetic concentration. Anesthesia & Analgesia 97(September):718-740. Abstract available at http://www.anesthesia-analgesia.org/cgi/content/abstract/97/3/718.

Xi, J. . . . and R.G. Eckenhoff. 2004. Inhalational anesthetic-binding proteins in rat neuronal membranes. Journal of Biological Chemistry 279(May 7):19628-19633. Abstract available at http://www.jbc.org/cgi/content/abstract/279/19/19628.

Further Readings:

Campagna, J.A., K.W. Miller, and S.A. Forman. 2003. Mechanisms of actions of inhaled anesthetics. New England Journal of Medicine 348(May 22):2110-2124. Extract available at http://content.nejm.org/cgi/content/extract/348/21/2110.

Franks, N.P., and W.R. Lieb. 1999. Background K⁺ channels: An important target for volatile anesthetics? Nature Neuroscience 2(May):395-396.

Ishizawa, Y. . . . and R.G. Eckenhoff. 2002. G protein-coupled receptors as direct targets of inhaled anesthetics. Molecular Pharmacology 61(May):945-952. Available at http://molpharm.aspetjournals.org/cgi/content/full/61/5/945.

Sources:

Roderic G. Eckenhoff
Department of Anesthesia
University of Pennsylvania
Philadelphia, PA 19104

Alex S. Evers
Department of Anesthesiology
Washington University School of Medicine
660 South Euclid Avenue
Campus Box 8054
St. Louis, MO 63110

Nicholas P. Franks
Biophysics Section, Blackett Laboratory
Imperial College of Science, Technology and Medicine
Prince Consort Road
London SW7 2BZ
United Kingdom

Neil L. Harrison
Laboratory of Molecular Neuropharmacology
Department of Anesthesiology
Weill Medical College of Cornell University
New York, NY 10021

Uwe Rudolph
Institute of Pharmacology and Toxicology
University of Zürich
Winterthurerstrasse 190
CH-8057 Zürich
Switzerland

James M. Sonner
Department of Anesthesia
S-455
University of California, San Francisco
San Francisco, CA 94142-0464

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