A
Study of Gamma Lactones and Their Ability to Antagonize Cocaine’s Effects in
Planarians and the Consequential Change in the Expression of Dopaminergic
Membrane Proteins
Debra
Baker
Department
of Biology
West
Chester University
M.S.
Thesis
Table
of Contents
Introduction
…………………………………………………………………………. ….. 6
Information
about Planarians ……………………………………………………………..6
Dopamine
………………………………………………………………………………...7
Cocaine’s
Effect on the CNS ……………………………………………………………..8
Information
about Cocaine ……………………………………………………………….9
Cocaine
as a Local Anesthetic …………………………………………………..10
The
Evolution of the CNS……………………………………………………………….13
Parthenolide
and Gamma Lactones……………………………………………………..16
Planarians
as a Model Organism…………………………………………………………17
Previous
Research using Planarians ……………………………………………………..19
The
Use of DMSO as a Solvent………………………………………………… 20
Testing
Sesquiterpenes ………………………………………………………………….21
Part One …………………………………………………………………………………………24
Methods
and Materials………………………………………………………………….. 26
General
Procedure ………………………………………………………………………26
Surface
Area to Volume Ratio ………………………………………………………….30
Results……………………………………………………………………………………31
The
Effect of γ-Lactones on Planarian Motility…………………………………31
Discussion
………………………………………………………………………………34
Similar
Results in Mammals…………………………………………………………….
35
Part Two
…………………………………………………………………………………………36
Introduction
…….………………………………………………………………………. 37
Methods
………………………………………………………………………………….38
Procedure………………………………………………………………………………...39
Analysis
of Results………………………………………………………………………42
Expected
Outcomes ……………………………………………………………………..43
Alternative
Approaches …………………………………………………………………46
Part Three
……………………………………………………………………………………… 47
Introduction
……………………………………………………………………………...48
Methods
and Materials …………………………………………………………………..48
Substances
Tested ……………………………………………………………………….50
Expected
Outcomes/Alternative Approaches ………………………………………….. 51
Room
for Improvement …………………………………………………………………51
Part Four………………………………………………………………………………………XX
Introduction……………………………………………………………………………XX
Materials………………………………………………………………………………XX
Experimental Design……………………………………………………………………XX
Experimental Design for
NMDA………………………………………………XX
Experimental Design for
Cytisine………………………………………………XX
Results………………………………………………………………………………….XX
Discussion………………………………………………………………………………XX
Room for Improvement…………………………………………………………………XX
Future
Direction………………………………………………………………………XXx
Bibliography
………………………………………………………….…………………………52
Appendix…………………………………………………………………………………………57
Published Papers Based
Upon This Work……………………………………………………XX
“Science
and research, and technology are like treasure.” Hannah started warming to her
favorite subject and glad to have a receptive audience, “Its lying there,
hidden in plain sight, ready to be discovered, has *always* existed but not yet
known. We are fortunate to live in the right time and place and given the
opportunity to discover it” She continued, “The heart of a person guides
actions with intention. Radiation has been used to kill people but radiation
has also been used to heal; without love we are noise but with love…”
“Love
is the currency of Heaven,” continued Anna thinking of Miss Viola.
Acknowledgements
I
thank God for the gift of life and for my unique temperament
I
thank my parents, Joan Sander and James Sander, for doing the best they could
with what they were given.
I
want to thank my husband, Pat, for supporting me
Also
my children, Jessica, Jennifer, Joanna, Jonathan, Jeanette, Joshua, Joseph, and
Julianna, who inspire me with their own uniqueness
And
my granddaughter, Fiona, who was born as this was being written and all of her future
siblings and cousins
Dr
Gestl, who put up with endless questions
Dr.
Mbuy, who encouraged creative thought and teaches by provoking learning
Dr.Pagan,
who told me to believe in myself.
Sean
Deats, who met me in the middle of the Biology-Psychology bridge; thank you for
brainstorming with me.
All
my lab partners, Sean, Peter, Daniel, Matthew, Erica, Clinita, Dharini, and
Galina; Whenever I remember you it will include laughter.
Introduction
and
Background
Information
Introduction
Planarian
worms are being used as model organisms in research related to Neuroscience because
they occupy a unique niche in the evolution of the Central Nervous System. They
are believed to be the extant species that is the closest, morphologically, to
the common ancestor shared by all subsequent species that possess a centralized
nervous system. The planarian nervous
system is relatively simple but, paradoxically, it possesses the same neuronal
elements that are found in more evolved species. Genes of the central nervous system are very
highly conserved. Consequentially, there
is reason to believe results obtained by observing planarians have the
potential to contribute to a better understanding of similar processes in
mammalian species including humans. Because the planarian genome has been
sequenced and resulting data are being made available, observing and analyzing behavior
in planaria has become an attractive subject for investigation.
Another
quality that is expressed intensely in planarians is an extraordinary ability
to regenerate. Planarians have the highest concentration of Pluripotent stem
cells found in adult organisms. The
potential applications using stem cells is just beginning to be explored. Because planarians are both able to respond
to psychoactive substances in a quantifiable and predictable manner and they
are able to regenerate any organ in their system including the cerebral
ganglion, also known as a brain, we have been able to determine the onset of
function in the planarian brain that is necessary to evoke a response upon
exposure to cocaine and other substances that act upon the nervous system.
Planaria is the common name for several genera
of free-living non-parasitic flatworms. Taxonomically, planarians belong to the
phylum Platyhelminthes, class Turbellaria, order Tricladida (Carranza,
et al. 1998). Planarians make up various
family, genera, and species. Although
they possess three germ layers, planarians are acoelomates.
Planarian Biologists in Asian labs tend
to work with Dugesia japonica. As its name implies, it is a common species of
planaria in Japan. Dugesia tigrina is the most common species in the United States but
it is the genome of Schmidtea
mediterranea has
been sequenced by Dr. Alejandro Sánchez Alvarado
and his colleagues at the University of Utah. The accomplishment of the Sanchez
lab opens up access to genetic assays that will enable biologists to determine
transcription and expressing rate while siRNA will be used to silence genes to
determine function (Gentile, et al., 2011; Sofia, 2007). It is
not unreasonable to predict S.
mediterranea will replace both D.
tigrina and D. japonica in the
next five years (Gentile, et al., 2011).
Planarians occupy an auspicious position
in the evolution of the central nervous system the details of which can be
found on page cite.
Although their system is simple, they possess features that suggest a
pivotal place in evolution They are closely related to the first organism to
evolve a brain and share pivotal central nervous system genes with almost every
contemporary organism that possess a CNS (cite source.) Planarians process and dispose of nitrogenous
waste using flame cells. Recently, the physiology
of flame cells has been studied and these cells form a prototype of a nephron
(Need citation.)
Planarians are triclad but do
not possess a coelem(get spelling) and do not have an alimentary canal. They access food using an appendage called a
proboscis. Solid waste exits the body via the proboscis (cite.)
Most species of planarians
(including the ones used in this project) are hermaphroditic possessing both
male and female genitalia (cite.)
Information
about Dopamine
Figure 1: Dopamine Molecule
|
There are two major types of dopamine
receptors and planarians have both types. They are D1 (including D1 like) and
D2 (including D2 like.) The major difference between these two receptors is
that, upon stimulation, D1 receptors are coupled with the G-protein that
stimulates the release of adenylate cyclase (cAMP.) Conversely, when D2 receptors are stimulated,
the activity level of cAMP decreases or is unaffected (Venturini, et al.,
1989).
The dopamine transporter (DAT) is a
transmembrane protein that pumps dopamine out of the synapse and into the
neuron from which it originated (Venturin, et al., 1989). Dopamine is repackaged into vesicles to be
used again.
Dopamine can be modified by
Beta-hydroxylase and processed into norepinephrine. Because it was regarded
(correctly) as a precursor of norepinephrine and epinephrine, Dopamine was not
recognized as a neurotransmitter in its own right until the 1950’s (get source
for this factoid.)
Cocaine is an addictive drug as a result
of the way that it exploits the dopaminergic neural pathways, experienced as a
sense of euphoria in human subjects. Dopamine
is prevented from being cleared from the synapse when cocaine acts as a
competitive reuptake inhibitor. Eventually, more cocaine is needed for the
addict to experience the same intensity of euphoria. Recently, knockout mice without dopamine
transporters have demonstrated addictive “seeking” behaviors which led to the
discovery that cocaine affected serotonin transporters as well as dopamine transporters
(Ribeiro, et al., 2005; Iversen, 2009; and
NIH.1998).
Because dopamine and serotonin are both
associated with motivation, pleasure, and reward, cocaine is an extremely
addictive substance. The dopaminergic system is actually altered in chronic addicts
(Volkow, et al., 1993). Because cocaine
blocks the dopamine transporter, and excessive dopamine accumulates in the
synapse, the number of postsynaptic receptors is actually decreased in
long-term addicts (NIH, 1998).
Additionally, some physiological effects
of cocaine exposure include tachycardia, elevated body temperature, decreased appetite, and an increase in available
energy; the result of cocaine’s effect on epinephrine. (Koehntop, et
al., 1977)
Cocaine is a derivative of the coca
plant, Erythroxylum coca, which
thrives in its woodland environment of South America. There are actually
several species of shrub that are in the Erythroxylum Genus but the species
most used and most associated with cocaine is Erythroxylum coca.
Indigenous
peoples of that region have taken advantage of the medicinal properties coca
leaf and have also incorporated it into their religious rituals. Generally,
indigenous peoples struck a balance with their natural environment and used the
coca leaves with respect and moderation (Personal
conversation with Sue Mustalisch, Professor of Health Science at West Chester
University). There have been no documented cases of addiction as the result of
using coca leaves (find source.)
According to anthropologists, natives of
Peru were aware of the anesthetic properties of the coca plant. They would chew
the leaves and drool on the part of the body that needed to be cut. Herbal treatments
derived from coca leaves were applied to the wounds associated with trepanning procedures
that were performed into the early part of the twentieth century (Musto, 1991).
There are some accounts of the
indigenous use of white powder and herbs that would increase energy and take
away painful sensation. These accounts were documented by the priests and
scribes that accompanied the Spanish in the fifteenth and sixteenth century
(Calatayud, and Gonzalez 2005).
Cocaine was initially transported to
Europe and studied by Friedrich Gaedcke. Starting in 1855, he extracted an “amorphous
substance” from the coca plant and identified it as an alkaloid naming it Erythroxyline. In 1860, another
chemist, Dr. Carl Sherzer, transported cocaine to Europe and,
along with his colleague, Albert Niemann, extracted cocaine from Erythoxyline. Niemann was the first European known to
observe the anesthetic effect of cocaine when it numbed his tongue (Koller,
1941).
Figure 2: Cocaine Molecule
|
Cocaine
was the first clinically used local anesthetic
(Sholz, 2002). Albert Niemann isolated
cocaine from the coca leaves (Koller, 1941) and Sigmund Freud was
the first one to treat a patient with it. Ironically, Freud used cocaine to
wean his patients from morphine. He and colleague, Karl Koller, were also the
first to demonstrate its numbing effect when applied occularly. A conundrum exists in this narrative that may
lead the reader to believe that two men were given credit for discovering
cocaine as a local anesthetic. Although Koller cited Anrep’s previous work
that was published, Koller was credited with the discovery because his work
that confirmed Anrep’s original findings was published in a professional
journal that was prominent to the surgeons of his era (Yentis and Vlassakov,
1999) Dr. William Stewart Halsted is
credited to be the first to describe the effect when cocaine is injected into
the sensory nerve trunk (Osborne, 2007).
Unfortunately, no one knew about the addictive qualities of cocaine.
Freud, as well as many other prominent people of the late Victorian era, became
cocaine addicts before laws could be passed to regulate the
substance (Koller, 1941).
The
mechanism of action that interferes with the transmission of sensation is a
disturbance in the resting potential in the sodium gated ion channels. Most theories involve open/closed,
active/inactive channels. Other theories propose a disturbance in the
phospholipid bilayer of the cell membrane. Local anesthetics are generally divided into
amides and esters (Vandam, 1987).
Amylocaine
was the first synthetic local anesthetic. It was synthesized in 1903 and was
quickly overshadowed by the formulation of procaine in 1904. Both Amylocaine
and Procaine (Novacaine) are esters and analogs of cocaine (McLure and Rubin,
2005). The slight change in molecular conformation
was sufficient to lose the addictive properties associated with cocaine while
preserving its effectiveness as a local anesthetic. Soon after Procaine was
first synthesized in 1904, it became widely available in dental offices and
emergency rooms. During the second half of the twentieth
century, Amides took the place of esters and local anesthetics took effect more
rapidly and had a lower incidence of allergic reactions (Calatayud and Gonzalez,
2005).
From the plant’s
perspective, cocaine evolved as a potent weapon in E.coca’s botanical arsenal. Cocaine affords effective protection
against predation. It is worth mentioning the likelihood that many potential
future pharmaceuticals may be found in the arsenal of hundreds
of thousands of known and yet-to-be discovered plants, animals, and microscopic
life forms that are players in the evolutionary arms race that share habitat
with Erythroxylum coca (Perfecto and Vandermeer,
2008). The cocaine molecule has four chiral centers which allow it to form 16 isomers
(McLure and Rubin, 2005).
Parthenolide and Gamma Lactones
Figure 3: Parthenolide
|
Determining any changes in the
transcription rate of key components in the cocaine-sensitive dopaminergic
pathway will contribute to a more comprehensive understanding of the mechanism
of action that lactones employ as cocaine antagonists.
In one sense, parthenolide was
an obvious choice A detailed explanation of the reasoning behind choosing
parthenolide can be found on page (fill in.) A few roadblocks exist chief among
them is the very low solubility of parthenolide. There is some effort on the
part of chemists to synthesize an analog of parthenolide with high solubility
Gamma Lactones were not selected
randomly for examination. When several
compounds were introduced to the aqueous environment in which planarians were
contained, the compounds with the five-carbon lactone ring structure were found
to alleviate the withdrawal symptoms in planarians with Parthenolide
demonstrating the greatest effect (Pagán, et al., 2008). Parthenolide is similar in many ways to
compounds such as β-eudesmol, which
shares similarities in molecular structure that had no significant effect on
planarians that were exposed to cocaine. The structures that were active often
had the lactone structure (a“lactone” is a ringed ester.) These ringed structures can also be found in many
traditional herbal medicines that have been used by people for thousands of
years. Feverfew (Tanacetum parthenium) is a good example.
Parthenolide is one of the identified molecules in this herbal remedy (Erlich,
2008).
The
Evolution of the CNS
Development of
bilateral symmetry and cephalization were important precursors to the evolution
of the Central Nervous System (CNS). Bilaterians most likely descended from a
common flatworm-like ancestor during the Cambrian geological period
approximately 550-600 million years ago (Jacobs, et al., 2005).
Until a few years ago, the dominant theory was
that vertebrates, insects, and flatworms diverged from a common bilaterians
ancestor, appropriately named Urbilateria, and went on to evolve giving rise to
three distinct nervous systems; the ganglion and ladder-like nerve cord of planarians,
the segmented ganglia of arthropods, and the brain and spinal cord and inverted
dorsoventral axis of vertebrate systems, (Sarnat and Netsky, 1985). New evidence suggests the Urbilateria had not
only developed bilateralism but also acquired the genes that form the
foundation of a CNS (Jacobs, et al. 2005). All organisms with a defined CNS
have origins in this common ancestor.
This is reflected in the evidence that many associated genes are
extremely highly conserved across all species that possess a CNS (Mineta, et
al., 2003). Although significant differences
exist in the morphology of individual species, each possesses corresponding
regions that, even when their neurological systems are morphologically
dissimilar, posses, “similar molecular fingerprints,” that reflects a high
degree of conservation (Sarnat and Netsky, 2002).
Figure 5:Map of Planarian Brain
Regions. Picture Courtesy of K. Agata.
|
A large
commissure crosses medially uniting planarian hemispheres reminiscent of a
mammalian corpus callosum (Sarnat and Netsky, 1985). The planarian ganglion
is, indeed, considered a primitive brain. The simple representation in Figure 5 shows
bilateralism with the yellow regions being devoted to mechano-sensory
activities, the green are chemosensory, blue represents light-sensory, and red
represents inter-neuron activity (Agata, 1998).
Planarians also possess a peripheral nervous system that is distinct
from the central nervous system. The planarian cephalic ganglion exhibits many
morphological features similar to the vertebrate CNS, such as multipolarized neurons.
The ratio of brain size to body mass in planarians is similar
to rats and young planarians have a higher ratio compared to adults. This is
similar to mammalian species but is not a feature in other simple animal
examples (cite source.)
A consequence of evolving a central nervous system is the emergence
of special sense organs and these are represented in simple presumably newly
evolved form in the planarian brain. The eyespots are not eyes as mammals
experience eyes but are organs able to sense light from dark. The auricles are
associated with hearing but in planarians, they are not quite hearing organs.
They have ciliated neurons that resemble olfactory bulbs and are actually chemo
receptors. Some species of planarians
have a statocyst which is a simple vestibular organ (Sarnat and Netsky, 1985).
Taxonomically,
planarians belong to the phylum Platyhelminthes.
The taxonomic niches are being adapted by molecular
analyses of the 18s rRNA sequences and, using this measure, bilateral
animals are being classified into three groups: Deuterostomia, Ecdysozoa, and Lophotrochozoa
(Huson and Scornavacca, 2011). Platyhelminthes
are found near the root of Lophotrochozoa.
Because planarians are so close to the
morphology of the early bilateral ancestor and because planarians occupy a
unique place wherein they possess qualities shared by vertebrates, they are an
ideal species to use in the study of the evolution of the CNS (Agata, et al.,
1998)
Key Genes for
the CNS and Their Conservation Across Species
Figure 6: Source K. Agata
Figure
6: Key Genes for the CNS
When key genes in the
central nervous system were compared across species, 110 of 116 head genes
found in planarians were shared by all of the bilateral animals that have been
examined (C. elegans, D. melanogaster,
and H. sapiens.) The absent genes ,
the 6 out of 116 genes that were analyzed, were absent in C. elegans and/or in D.
melanogaster. These three species belong to the three different groups of
bilaterians that have a CNS (Agata, et al., 1998). These genes include FGF signaling molecules,
noggin, and frizzled, among many others.
Because so many functional genes are so highly conserved, one leading
theory suggests that all bilaterians with a CNS share a common ancestor (Sarnat
and Netsky, 1985).
Figure
7: Bilateral Symmetry in Planarians
Because the CNS seems
to be so highly conserved and because planarians appear to be the closest
extant organism to a shared common ancestor, several research laboratories have
been busy observing and documenting normal behavioral patterns in planarians
that have been exposed to various chemicals that act upon various
neurotransmission pathways. Responses to
compounds such as cocaine (Raffa, et al., 2003), caffeine (Pagan, et al.,
2009), nicotine, (Pagan, et al., 2009), aspartame (unpublished data, Baker,
2010), and NMDA (Rawls, et al., 2007), have been observed in planarians. These observations have established a variety
of behaviors that are considered normal which has, in turn, made it possible to
identify behavior that departs from the standard (Raffa, et al., 2001).
With respect to
cocaine, planarians exhibit predictable behavior patterns upon exposure to
cocaine at sufficient concentrations.
Their movement is retarded significantly. They also go through
seizure-like movements during which they frequently curl up into a, “C-like
position.” This is a spasm-like behavior
that is distinguishable from the more smooth movements that are within the
planarians repertoire. Another behavior
that is associated with cocaine toxicity is the, “head bop,” in which they
appear to have the tail pinned to the bottom of the dish while they waggle back
and forth (Raffa, et al., 2001; Pagán, et al., 2008).
Planarians
as a Model Organism
By the turn of the
twentieth century, Biologists were aware of the regenerative abilities of
planarians. The earliest known reference
was found in a Japanese encyclopedia dating to the seventeenth century
(Sanchez, 2010). Over 600 scholarly
papers about planarians were published around the turn of the twentieth century. Research varied from light-dark studies to
regeneration after exposure to radiation.
The reason planarians
stopped being used in advanced research was a direct result of the intellectual
rivalry between two distinguished developmental and genetic biologists, namely
Charles Manning Child and Thomas Hunt Morgan (Clark, 1992).
Thomas Hunt Morgan
embraced Mendelian Inheritance theories and believed there was a distinction
between somatic and germ line genetics. Since Drosophila melanogaster expresses its germ line earlier in development,
D. melanogaster became a model organism for use in developmental and
genetics research (Nobel Prize Biography).
Child used planarians
in his research and was much more interested in environmental influences and
phenotypes. Child was not quite convinced that genes were stored
on chromosomes. He also rejected the
notion that two discrete sets of molecular instructions exist; the somatic and germ
line (Clark, A., 1992).
Child and Morgan chose
to ignore one another’s existence and certainly did not acknowledge the other’s
contribution to genetics and developmental biology.
Eventually, Morgan’s
ideas were supported by subsequent discoveries and his work with D. melanogaster
became associated with the emerging discipline while planarian research fell
out of favor (Clark, 1992). Ironically, some trends have come full circle and
the work Morgan pioneered in the field of genetics has resulted in the mapping
of the human genome as well as the genomes of many other species. As a result, the focus of research has
shifted to epigenetics; the study of how environment interacts with the basic
instructions contained within the cell, and stem cell technology that holds the
hope of innovative therapies. Because
they have adult pluripotent stem cells distributed throughout their body, planarians
are able to allow regeneration of an entire individual from a tiny fragment of
tissue. As a consequence of their remarkable ability to regenerate and because
of their status as the least evolved organism with a central nervous system, planarians
are being rediscovered as model organisms in labs all around the world (Gentile,
2011).
Advancements in
sequencing DNA have contributed to resurgence in planarian Biology. Dr. Alejandro Sánchez Alvarado was among the first to
recognize the potential benefits of sequencing the genome of at least one
species of planarians. It was necessary for him to harvest planarians from a
fountain in Spain. The genus and species of his planarians just happened to be,
“Schmidtea mediterranea,” which is why
this particular genus and species will most likely emerge as the model species
for planarian research into regeneration (Newmark and Sánchez-Alvarado, 2003).
Planarians are emerging
as model organisms in the areas of regeneration research, particularly using
adult stem cells and in the area of neurological and neuropharmacological
research (Gentile, et al., 2011). Along with the ability to regenerate and their position on
the evolutionary clade that places them as the most anciently evolved species
with a centralized nervous system and rudimentary special sensory organs,
planarians are able to reproduce sexually and asexually. S. mediterranea houses its
genome on four stable diploid chromosomes (Lau, et al., 2007).
The lack of a stable genome
within the Dugesia genus will adversely affect the ability to conduct the tests
necessary to discern what is happening to the transcription rate of the
associated RNA and subsequent changes to the expressing level of key protein
products.
The
Use of DMSO as a Solvent
Figure 8: Diethyl Sulfoxide (DMSO)
|
Testing
Sesquiterpenes
Various organic
compounds have been tested to determine if any of them was able to antagonize
cocaine’s effect in planarians.
Eventually, a group of ringed organic Sesquiterpenes were suspected to
be good candidates for investigation. After careful observation, a Sesquiterpene
with the five sided gamma lactone, Parthenolide, was found to antagonize the
symptoms of cocaine’s activity in planarians (Pagan, et al., 2008).
The idea of testing Parthenolide against
cocaine in planarians originated in Dr. One Pagan’s dissertation. The following
narrative is a direct quote taken from Dr. Pagan’s dissertation defense.
“Why parthenolide? The idea of using parthenolide as a
cocaine-displacing ligand came from its close structural similarity to another
class of natural products, the cembranolides.
Cembranolides are cyclic diterpenoids widely distributed in nature. More than 300 examples have been described
(Wahlberg & Eklund 1992).
The
cembranolides
are established ligands of the nicotinic acetylcholine receptor (nAChR). The nAChR is the best-known member of the
ligand-gated ion channel superfamily.
This receptor will not be discussed here, and the reader is referred
elsewhere (Karlin, 2002). Cembranolides
bind in a mutually-exclusive manner with the local anesthetic procaine, on the
nAChR (Pagán, 1998; Pagán et al., 2001).
The
“line of thought” that led me to consider parthenolide as a cocaine antagonist
was:
1. Cembranolides and an example of a local
anesthetic bind to nicotinic acetylcholine receptors (nAChRs) in a mutually-exclusive
manner (Pagán, 1998, Pagán et al., 2001).
2. Cocaine is the prototype of the local
anesthetic class of molecules, with neuronal sodium channels as the target
(Ruetsch et al., 2001; Scholz, 2002).
However, its behavioral effects are due to cocaine’s interaction with
neurotransmitter transporters, mainly the dopamine transporter (Uhl et al.,
2002).
3. Parthenolide shows close structural
similarity with cembranolides.
4. Parthenolide interacts with nAChRs and is
capable of alleviating cocaine inhibition of this receptor in
electrophysiological studies (Pagán and Hess, unpublished results).
Based on the points above, I thought
that parthenolide and cocaine had the potential to interact with the dopamine
transporter” (Pagan, 2005).
Dr. Pagan’s
educated hunch seems to have served him well as yet-to-be published data that
is the Master’s thesis of one of his graduate students strongly supports the
hypothesis articulated in Dr. Pagan’s dissertation. Thomas M. Graczyk, MS, has transfected
dopamine transporter genes (DAT) into embryonic kidney cells that normally do
not possess dopamine transporters. He
then exposed these cell lines to procaine (an analog of cocaine) in the
presence and absence of parthenolide and the parthenolide reverses the effect
procaine has upon the DAT. His experimental design included negative and
positive controls one of which allowed for repeated tests in cloned cell lines
that did not possess DAT. These non-DAT lines did not see the same effects
(cite thesis as source.) One of the more intriguing results of this endeavor
includes the discovery that the binding site of parthenolide to the dopamine
transporter is allosteric and non-competative with dopamine or cocaine. It apparently increases the affinity for dopamine.
Up until this point, the model for the binding site cocaine occupied to
antagonize movement and increase seizure-like movements was thought to be both
orthosteric and competitive with dopamine at the transporter site (cite.)
Cocaine ester acts as
an anesthetic as well as a dopamine reuptake antagonist (Koehntop, 1977).
Cocaine amide acts as a local anesthetic but does not produce the behavioral
effects that are associated with excessive dopamine levels (Iversen, 2009).
Figure 9; Parthenolide (left) and
Euniolide (right) Shown together to illustrate structural similarity.
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