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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5515_Библиотеки_им_академика_М_И_Перельмана.pdf
As with most original thinking in Western humans, there was not much progress
beyond Aristotle and his students. Except for some novel and important work in the
Arab world and the Far East, a sort of conceptual dead zone occurred for almost two
millennia until a great age of anatomical discovery and analysis started at the end of the
1700s and early 1800s, when organs and organ function were explored extensively. At
the end of the eighteenth century, Félix Vicq d’Azyr, a French medical specialist,
suggested there were nine general properties of life. These included digestion,
circulation, breathing, and reproduction, which are now associated with our digestive
system, our circulatory system, our respiratory system, and our reproductive system.
Over the next few centuries, other organ systems were added to the list. Perhaps two of
the most important to be added during this time were the nervous system and the immune
system. Study of these two systems led to a deeper understanding of the molecular
phenomena involved in body systems.
Counting organs, organ systems, and body systems might seem to be an esoteric
task. But oddly, scientists have settled on ten or eleven major organ systems in the
human body. These systems are skeletal, muscular, nervous, endocrine, cardiovascular,
lymphatic, respiratory, digestive, urinary, and reproductive. This list ignores skin (the
eleventh system in an eleven-system scheme). Given that our exterior integument is one
of the largest organs in our bodies, this could be viewed as a major oversight, but it
also points to the difficulty of recognizing the various parts of our bodies as integrated
systems.
There is, however, one further system that has been a latecomer to the fray. The
synthesis of THC in the 1960s led to an entirely new area of brain science in human
biology. It was inevitable that discovery of this last system occurred so late. THC was
the trigger for the discovery of this system, and research into an entire body system was
stymied because of the illegal status of cannabis. The story of the discovery of this
system actually starts with another category of controlled substances: the opioids. In
1973 researchers determined that the small opioid molecules such as heroin and
morphine would bind to specific areas of the brain. Johns Hopkins University
researchers discovered that there were receptors for these opioids in the brain. Making
the leap from opioid receptors to receptors for the active ingredient in marijuana was
simple. But it took fifteen years to document their existence conclusively, when
researchers at St. Louis University School of Medicine showed that the brain had
receptor molecules embedded in the membranes of nerve cells that recognize analogs of
THC. Allyn Howlett and William Devane were the scientists who pinned this down,
and they further showed that these receptors were much more ubiquitous in the brain
than any of the other receptors previously examined, such as acetylcholine, endorphin,
serotonin, glutamate, or gamma-aminobutyric acid (GABA) receptors.
The next question was where in the body these receptors resided. Researchers
knew that the brain harbored many of them, but suspected the receptors for THC were
also elsewhere in the body. In the 1980s the Pfizer pharmaceutical company was able to
synthesize an efficient THC analog that could be labeled with radioactivity and injected
into the body. By detecting where the radioactivity ended up, presumably bound to
natural receptors, researchers could also determine where the receptors for this
cannabinoid existed. To their surprise the receptor sites were distributed throughout the
body, making this system more widespread than just the brain. In 1990 pharmacologist
Lisa Matsuda and her colleagues announced the cloning and sequencing of the gene for
cannabinoid receptor molecule from a rat brain. Thirty years after the chemical
characterization of THC, researchers had pinpointed the receptor for this small
cannabinoid molecule. The receptor molecule Matsuda and colleagues characterized
was called the CB receptor, and its sequence was similar to a category of proteins
being rapidly discovered at the time called G-coupled protein receptors (GCPRs). They
are all recognizable because they reside in cell membranes and have seven highly
characteristic helices that wind back and forth through the membrane, with one end of
the protein extending into the cell and one end sticking outside. Three years later
molecular biologist Sean Munro and colleagues cloned and characterized another
molecule that served as a receptor found in the spleen. Since this was the second CB
receptor found, the original CB receptor became CB-1 and the new one was named CB-
2.
The story wasn’t yet complete, however. Researchers had found the receptors that
bound THC, an exogenous cannabinoid. Certainly if these receptors existed, then they
had to have some interaction with an endogenous molecule or multiple molecules that
were part of the body’s normal functioning. In 1992 a group of pioneering researchers
isolated and characterized this endogenous factor: an endocannabinoid that they named
anandamide (AEA). Three years later they discovered and characterized a second
endocannabinoid called 2-arachidonoylglycerol, or 2-AG. The differences between
AEA and 2-AG are discussed in detail below. Because of its ubiquity in the human
body and the importance of its function, this receptor—ligand system was named the
“endocannabinoid system.”
“I Have Never Used It”
How do we know so much about cannabinoids and the endocannabinoid system?
Perhaps primarily because of one man: Raphael Mechoulam. During the writing of this
book, this amazing scientist passed away at the age of ninety-two. He remained active
as a scientist until his death on March 9, 2023. Some call him the father of
cannabinoid/endocannabinoid research; he is best known for discovering THC in 1963
and the primary endocannabinoid of our brains—anandamide—in 1990. (“Discovering”
means the first to isolate the compound and determine its structure and chemistry.) His
early life included hiding from Bulgarian Nazis during World War II and immigration in
1949 to Israel, where he did outstanding chemistry research. When he was appointed to
his first professorship in the early 1960s at the Hebrew University of Jerusalem, he
scoured the literature for a study system that would offer long-term research
opportunities. Being trained in the chemistry of natural compounds, he focused on plant
systems where he might find a compound that could be used in chemistry or
pharmaceutical development. His literature search unveiled a strange observation.
Despite its important role in plant phytochemistry and in illicit drug trade, the active
chemicals of cannabis had little to no research focused on them. Here was the
intellectual gold mine he was seeking. But he quickly realized that the reason for the
lack of research on cannabis was probably the illicit, illegal, and punishable aspect of
possessing cannabis. Like any clever person, he found a work-around, as detailed in his
2023 memoir:
How does one get cannabis—a strictly regulated illicit drug—in sufficient amounts to initiate
research? In 1963! Again, I was lucky. The administrative head of my Institute knew a police
officer, who was presumably the number two (or possibly the number three) in the Israeli Police
hierarchy. He phoned and told him that a Dr. Raphael Mechoulam needed hashish for research
and that he—meaning me—was completely reliable (though he barely knew me). I just went to
Police headquarters, had a cup of coffee with the policeman in charge of the storage of illicit
drugs, and got 5 kg of confiscated hashish, presumably smuggled from Lebanon.
Given that another famous scientist, neurologist Oliver Sacks, had a penchant for
taking and experiencing his research drugs, I wondered whether Mechoulam partook of
the noble weed. In an interview for Culture magazine in 2017, he said, “I have never
used it.” This was after fifty-five years of research on the plant and its psychoactive
effects. Mechoulam pointed out that any departure from being completely legal about
using the hashish would have landed him in jail and, worse, would have destroyed his
chances of continuing work on the amazing plant.
Figure 11.1. Structure of anandamide and 2-AG.
His pioneering work on the endogenous cannabinoid called anandamide (from the
Sanskrit word ananda, meaning “bliss”) led to the better understanding of how our
bodies make large amounts of cannabinoids that move throughout our endocannabinoid
system. Anandamide (AEA) exerts an overall modulatory effect on the brain reward
circuitry and can bind to endocannabinoid receptors throughout the rest of the body to
suppress pain and tumors. Another endogenous cannabinoid is 2-arachidonoylglycerol
or 2-AG, which is found in higher concentrations in the endocannabinoid system than
AEA. There are other endocannabinoids such as noladin ether, palmitoylethanolamide
(PEA), virodhamine, and oleoylethanolamide (OEA) that our bodies make, but AEA
and 2-AG are the major players in the endocannabinoid system. Figure 11.1 shows the
chemical structure of these two major endocannabinoids.
The difference between anandamide and 2-AG are their “tails.” To me these
resemble the “centipede” triterpenes discussed in chapter 9. They are about the same
length and have a similar look as the exogenous cannabinoids THC and CBD. Even a
slight resemblance of an exogenous molecule to another active endogenous one will
cause confusion in the function of the endogenous one.
Where do we get these important neurotransmitters? They are synthesized from
genes in our genomes, and our bodies make a whole slew of enzymes that regulate the
production, storage, and release of these chemicals as we respond to physiological
challenges. The system of endocannabinoid synthesis and maintenance is quite complex,
involving tens of different enzymes and proteins. It is in and of itself a mini-kluge, as
these proteins work in concert in convoluted ways to regulate the cellular activity of
endocannabinoids. A diagram of the endocannabinoid system in a cell might make sense
to someone working on the system, but to the lay eye it would look much like the Mouse
Trap game I played as a kid. Different parts of the cell have different biochemical
contraptions that take care of a step or two of the inner workings of the endocannabinoid
system. They are all connected and work together to regulate the endocannabinoid
system.
So far, we have examined two of the three major parts of the endocannabinoid
system (endocannabinoids and where they bind). The third part is perhaps the most
interesting: the receptors that recognize the endocannabinoid molecules and transfer the
recognition into information in nerve cells. There are two major receptors for
endocannabinoids, conveniently named CB-1 and CB-2. These two receptors reside in
the cell membrane of a cannabinoid-sensitive neuron. Since they are GPCRs, part of
their structure sticks out of the cell and into the synapse (if one is present). On the inside
end of these kinds of GPCRs is a structure that binds other proteins as described earlier.
When a cannabinoid receptor site on the synapse side of the membrane encounters
something it will bind to, it does so and produces a change in the overall structure of the
GPCR, which then produces an interaction with intracellular protein complexes and
induces signaling.
If you look closely at these two structures, you will see well-defined similarities.
Each of the two proteins has seven helical stretches. The placement of the helical
structures is in the same general positions in both proteins (fig. 11.2). There are some
slight differences between the two proteins, though, which make them behave differently
when confronted with endocannabinoids and exogenous cannabinoids. These structural
differences make the specificity of binding to and the responses of CB-1 different from
the responses of CB-2. And where these receptors are synthesized in our bodies makes
all the difference as to how these two receptors behave. Figure 11.3 shows a diagram of
the human body and where the two receptors predominate.
Although the position in the body of these two receptors overlaps considerably,
there is a general trend in how they are dispersed. CB-1 appears to be very brain
centralized in its distribution. It also appears to follow general tracts of the peripheral
nervous system. CB-2 on the other hand is truly widespread in the body but does show
centralization in peripheral organ systems primarily in locations where immune function
is important. As the figure implies the two receptors have very different bodily domains
of effect.
Figure 11.2. Ribbon models of CB-1 (left) and CB-2 (right). Note that there are seven helical parts of each
protein (these will span the membrane of the nerve cell) and out-of-cell and inside-the-cell ends (at top and
bottom of each figure). Adapted from Shao et al. (2016).
Evolutionary Shuffle
When did these receptors first arise, and how widely dispersed are they in the tree of
life? Because the endocannabinoid system is so important in humans, knowing where
and when it originated in animal evolution constitutes an essential cog in understanding
their function. One might expect the origin of the endocannabinoid system in animals and
the origin of cannabinoid synthesis in plants to be correlated. At least that would be a
good hypothesis to test. A good way to perform this experiment is to identify the genes
in the genomes of as many animals as possible for the cannabinoid receptors CB-1 and
CB-2, and then to investigate how those changed with time. This kind of approach can
also determine the animal group (or common ancestor) from which certain genes were
gained.
But this approach also begs the question of how a new gene is acquired. Some new
genes are obtained by organisms through horizontal transfer or “jumping” from one
species to another. This mode is probably not how most animals and plants make new
genes, but it does predominate in bacteria and archaea. Another mode is to simply
duplicate an existing gene and let natural selection mold the duplicated gene into
something quite different. This mode is a tenable one for the CB genes. Yet another
mode is more drastic, but it has been shown pivotal in animal genome evolution. This
mode involves duplicating the entire genome of an organism during reproduction, so that
instead of having four chromosomes for instance in the diploid stage, the organism now
has eight. The genes on the duplicated four chromosomes are then “free” to diverge and
attain novel functions. This mode is particularly tenable for accruing new genes for
animals and plants; researchers have determined that full-scale genome duplications
have occurred in the evolution of several eukaryotic lineages. Multiple genome
duplications have occurred in plants and fish. One specific entire genome duplication
that has been identified in the common ancestor of vertebrates has been touted as an
important source for novel genes that led to the diversification of vertebrates.
Figure 11.3. Locations of CB receptors and their various functions. Adapted from Muralidhar, Maurya, and
Velmurugan (2019).
John McPartland, along with his colleagues Isabel Matias, Vincenzo Di Marzo, and
Michelle Glass, tested this hypothesis one step better in 2006. They examined not only
the distribution of CB-1 and CB-2 in the animal tree of life, but also an array of other
gene products involved in the synthesis of AEA and 2AG endocannabinoids. These are
the enzymes that make up the endocannabinoid system mini-kluge described earlier.
McPartland and colleagues’ studies were undertaken when there were only about a
dozen full genomes of organisms that had been sequenced. McPartland and his
colleagues searched the sequence database of each of these twelve genomes (a human, a
mouse, a tunicate, an apicomplexan, a ciliate, a fish, an insect, a nematode, a fungus, a
plant, an archaean, and a bacterium) for the genes that code for ten of the proteins
essential to the endocannabinoid system. Once these genes were found, a gene family
for each one could be estimated using phylogenetic methods. By examining the family
trees, one could then determine which groups have which genes.
The collection of twelve species in McPartland’s analyses is not a perfect
representation for a complete tree of life (many major groups are missing), but it will
suffice to trace the working parts of the mini-kluge that the endocannabinoid system
eventually became. Because this kind of research is what I do for a living, I have spent
some time repeating this experiment with the thousands of organismal genomes that are
now available; McPartland and his colleagues got it largely right working with just
these twelve species. Figure 11.4 shows a tree of life and the origin of each of the ten
genes used by McPartland and colleagues.
It should be obvious from figure 11.4 that the endocannabinoid system did not just
pop out of thin air, fully functional. If it had, then all the alphabet soup names of
enzymes would appear on only one branch of the diagram. Instead the endocannabinoid
system was molded in steps over millions if not billions of years of the evolutionary
process. For instance, the fatty acid amide hydrolase (FAAH) gene that catabolizes
AEA arose in the earliest of eukaryotes. But wait: there probably was no AEA being
produced in these early eukaryotes that lived about 1 to 2 billion years ago, because the
enzyme responsible for synthesizing AEA did not arise until the ancestor of fungi and
animals. (Yes, fungi and animals have a more recent common ancestor than plants and
fungi; in other words, a mushroom is more closely related to us than it is to a
sunflower.) Almost assuredly, fatty acid amide hydrolase arose as part of a completely
unrelated processing pathway and was “co-opted” by the AEA pathway. Another
example of the stepwise construction of the AEA pathway is the production of 2-AG.
This part of the pathway probably arose as a two-step process with DAGLα arising in
the common ancestor of all animals, and DAGLβ arising in the common ancestor of all
vertebrates. A final example concerns the finishing touches in the endocannabinoid
system. Remember that AEA is a neurotransmitter that bonds to other receptors. So what
it interacts with becomes an important part of the endocannabinoid system. One of these
receptor molecules is called the vanilloid receptor (TRPV1), and the second is a
receptor called GPR55. The genes responsible for synthesis of these receptors arose in
the common ancestor of mammals, indicating that once the full endocannabinoid system
came into existence in the common ancestor of vertebrates, the story of
neurotransmission wasn’t over. The various steps to the endocannabinoid system
happened at radically different times, with somewhat large gaps of time between the
steps.
Figure 11.4. Tree of life showing where the ten proteins arose in the endocannabinoid system.
A Delightful Trip
Let’s go on a “delightful trip” (Mechoulam used this phrase to describe his career in
endocannabinoid system study) and follow one endogenous cannabinoid molecule and
one exogenous cannabinoid molecule through a neural synapse. Let’s start with the
endogenous cannabinoid AEA, which first needs to be synthesized by our cells. A
clumsily named molecule NAPE-PLD (N-acyl phosphatidylethanolamine-specific
phospholipase D) synthesizes AEA from starting material called arachidonic acid. We
mostly obtain this precursor molecule from foods such as meat and eggs, and our cells
sequester it for use in synthesizing molecules that are important to the endocannabinoid
system like anandamide (AEA). The chemistry is rather complex, as our bodies can also
synthesize arachidonic acid, but the most reliable source of this molecule is through our
diet. Once the AEA is synthesized, it remains in the cell awaiting its use in the