INVESTIGATION OF THE EFFECTS OF
ANESTHETIC COMPOUNDS
ON LIPID BILAYER ORGANIZATION
By
Katherine L. Logan-Dinco

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
Chemistry - Master Of Science
2013

ABSTRACT
INVESTIGATION OF THE EFFECTS OF ANESTHETIC COMPOUNDS ON
LIPID BILAYER ORGANIZATION
By
Katherine L. Logan-Dinco
The present thinking in the scientific field about anesthetic action has evolved into two
hypotheses, both of which involve the cellular plasma membrane. The investigations presented
in this thesis focused on understanding interactions between model lipid bilayer structures and
three general anesthetics (pentobarbital, isoflurane and halothane) to determine the effect(s) of
direct interactions between bilayers and anesthetics. The synthetic vesicles used in this work
were intended to mimic qualitatively the neuronal membrane in lipid composition.

For

spectroscopic investigations, 0.5 – mole % perylene was added to the vesicle mixture prior to
vesicle formation. Fluorescence anisotropy decay measurements were used to investigate the
local environment formed by the lipid bilayer acyl chain region in the vesicles. The acyl chain
region of the vesicles was seen to undergo a change in the extent of the organization observed
with the control when exposed to selected anesthetics. These changes were observed with the
change in acyl chain viscosity. The change in local organization sensed by perylene rotational
diffusion was seen to be similar for all anesthetics used despite the absence of structural
similarities among the anesthetics. In all cases, a change in the organization of the lipid bilayer
was seen for anesthetic concentrations of ca. 4 mM. In all cases, the change in lipid organization
was observed to be an increase in the rate of rotational motion of perylene about the axis
perpendicular to its pi-system plane.

ACKNOWLEDGEMENTS
First and for most I would like to thank God for giving me the family, mentor, friends,
and guidance committee to take on such an accomplishment. Without the triumphs and struggles
He has helped me through it all or I would not be here today.
Next I want to thank my advisor, Gary Blanchard, for giving me a chance to learn
spectroscopy and making me care about it! I want to thank him for his guidance, patience,
understanding, and most of all for his faith in me especially when I was running low. Also, Gary
thank you for not letting me quit when the times got rough and for trying to learn more biology
so I could feel like I fit into the group.
My committee has been wonderful to me so thank you very much Dana Spence, Greg
Swain, and James Galligan. Thank you for your support in my educational growth as well as
research growth. Thank you for the guidance that you have given me. A special thanks to Dana
Spence for convincing me to apply to MSU and for being a friendly face that I can always ask
advice from!
I would like to thank my group, Doug Gornowich, Christine Hay, Iwan Setiawan, Chen
Qiu, Fredy Pratama and last but not least not only my lab mate but best friend Kimberly
Bostwick. I would also like to give a special thanks to our visiting student from Poland Krzysztof
Nawara. Thank you all for listening to so many presentations and for the advice you had shared.
Kim, thank you for being a great inspiration to me not only as a lab mate but as a friend. I could
have not done this without you girl!! You are one of the most amazing, well rounded, and
balanced people I have ever met and have had the pleasure to call a friend.

iii

Krzysztof, thank you for teaching me how to use the instruments, asking me how they work and
about my research all time to insure I was getting it right; you will make an excellent teacher one
day.
There were also so many people at State that helped me along the way through thick and
thin that deserve special mention here: Dr. A. Dan Jones, Joni Tucker, LeighAnn Jordan, Marion
France, Prabodha Ekanayaka and Cynthia Kaeser. Without them I would have been lost and
would have never made it through this process and for that I am thankful!
Last but not least my most supportive network my loving family. Mom and Dad thank
you for all the years teaching me the tool to be a strong independent woman. Thank you for the
love, support, discipline, guidance, and forgiveness. Theron, thank you for being the best little
brother in the world, for all the laughs, fights, and crazy times we have shared to get me to where
I am today . . . also thank you for proof reading! I would like to thank my in-laws for their
support in my studies and for my husband. Finally, I want to thank my husband, Dan, who has
been the backbone for me over the last two years and my best friend for the last nine years.
Thank you for the support and bearing the brunt of my emotions. I love you more than anything
in the world!

iv

TABLE OF CONTENTS
LIST OF TABLES………………………………………………………………………..…....…vi
LIST OF FIGURES……………………………………………………………………………..vii
LIST OF EQUATIONS…………………………………………………………………………..ix
CHAPTER 1………………………………………………………………………………………1
INTRODUCTION
CHAPTER 2……………………………………………………………………………………..28
EXPERIMENTAL PROCEDURES
CHAPTER 3……………………………………..………………………………………………36
RESULTS AND DISCUSSION
CHAPTER 4……………………………………………………………………………………..52
CONCLUSIONS
LITERATURE CITED………………………………………………………………………….54

v

LIST OF TABLES
Table 1.1: This table is a summary of the important properties of the inhaled anesthetics
halothane and isoflurane. MW - molecular weight, BP - boiling point, SVP - saturated vapor
pressure, MAC - minimal alveolar concentration, O/W - oil water, Bl/G - blood gas, W/G - water
[43]
gas, Br/G - brain gas, O/G - oil gas, Br/Bl brain blood interfaces
………………………….27
Table 3.1: Fluorescence lifetime and anisotropy decay time constants as a function of anesthetic
concentration. The fluorescence lifetime data were fitted to a single exponential decay function
and the anisotropy decay data were fitted to a two-exponential decay component
function………………………………………………………………………………………….43
Table 3.2: Dz, Dx, Dz/Dx, DSE and ηDSE values derived from experimental data as a function of
anesthetic concentration. …………………………………..…………………………………….44

vi

LIST OF FIGURES
Figure 1.1: Thiopental the first IV anesthetic discovered independently in the 1950’s by two
separate groups………………………………………………………………………………….... 3
Figure 1.2: A. Albuterol has 2 stereoisomers one of which causes undesirable side effects B.
Xopenex® contains the purified R isomer of albuterol………………………………………….8
Figure 1.3: Structures of A. Sevoflurane; B. Isoflurane; C. Thioental; D. Pentobarbital; E.
Nipectic…………………………………………………………………………………………..12
Figure 1.4: Structure of Enfurane………………………………………………………………..13
Figure 1.5: Propofol is a commonly used barbiturate…………………………………………...14
Figure 1.6: Refined structure of the nicotinic acetylcholine receptor at 4A resolution. Unwin, N.,
(2005) J.Mol.Biol.346: 967………………………………………………………………………15
Figure 1.7: A. DPH is a non-polar chromophore; B. TMA-DPH is polar……...………………..17
Figure 1.8: Structure of DPPC…………………………………………………………………...18
Figure 1.9: Left DPPE with an NBD chromophore tag, 1,2-dioleoyl-sn-glycero-3phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (ammonium salt). Right DPPA, 1,2dihexadecanoyl-sn-glycero-3-phosphate (sodium salt).…………………………………………21
Figure 1.10: Structure of Perylene……………………………………………………………...22
Figure 1.11: Structure of Cholesterol…………………………………………………………...22
Figure 1.12: Structure of Halothane……………………………………….…………………...27
Figure 2.1: Structure of Tris - HCl ®……………………………………………………………30
Figure 2.2: Representative schematic of TCSPC Instrument…………………………………....32

vii

Figure 2.3: Schematic of the principles behind the anisotropy decay measurement ..…………35
Figure 3.1. Left: Structure, excitation and emission spectra of perylene. Right: Structure of
18:1 NBD-PE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol4-yl) (ammonium salt)…………………………………………………………………………...37
Figure 3.2: A. How perylene would orientate in the Cartesian components if it were a prolate
rotor B. How perylene would orientate in the Cartesian components if it were an oblate rotor
………………………………………………………………………………………………… . 41
Figure 3.3: A. How perylene would orientate in the Cartesian components if it were a prolate
rotor, B. How perylene would orientate in the Cartesian components if it were an oblate rotor
…………………………………………………………………………………………………. . 45
Figure 3.4. Dependence of lipid bilayer acyl chain region viscosity, detected by rotational
diffusion measurements, on the identity and concentration of anesthetic present in the vesicle
containing solution……………………………………………………………………………….45
Figure 3.5. Top: Dz and Dx (GHz) as a function of pentobarbital concentration. Bottom:
Dz/Dx as a function of pentobarbital concentration……………………………………………..47
Figure 3.6. Top: Dz and Dx (GHz) as a function of isoflurane concentration. Bottom: Dz/Dx as
a function of isoflurane concentration…………………………………………………………...48
Figure 3.7. Top: Dz and Dx (GHz) as a function of halothane concentration. Bottom: Dz/Dx as
a function of halothane concentration……………………………………….…………………..49

viii

LIST OF EQUATIONS
……………………………………………………...……………..……...23

Equation 1.1 A =
Equation 1.2

..………………………………………..……..23

Equation 1.3

………………………….……………..…………………..……..24

(

Equation 1.4

)………………………………...……..…..………25
……………………………………………………………...…33

Equation 2.1

⁄

Equation 2.2

…………………………………………………...…………….….…33

Equation 2.3

( )

( )

(

Equation 2.4

( )

(
( )

Equation 2.5 R =

( )

( )

( )

( )) ………………………………………..……33
)…………………………………..………..……33

………………………………………………..…………..…...34

…………………………………….……………….35

Equation 2.6

Equation 3.1
………………………………………..………………..40
Equation 3.2

()

(

Equation 3.3

()

)……………………………………….….……...40

( (

)
ix

(

) ………41

Equation 3.4
………………………………………………….……………….42

Equation 3.5

(

)
……………………………………………….…42

x

CHAPTER 1
INTRODUCTION
Twenty to thirty million Americans undergo surgeries with the use of general anesthetics
every year for minor, major, and cosmetic surgeries.

1

Approximately 1 of every 13,000

individuals who undergo surgery dies due to complications related to anesthetic use.

2

These

fatalities can take many forms from cardiac arrest, pulmonary arrest, and drug overdoses. All of
these factors are caused by a reaction to the anesthetic being administered. While there are
factors that increase an individual’s risk of complications from surgery, such as obesity,
smoking, heart problems, and allergies, there is still a risk of undergoing general anesthetics for
surgery from the anesthetics themselves. General anesthetics are classified as organic agents
that, when delivered, cause a reversible loss of consciousness (awareness of surrounding) which
is defined by a combination of three states: analgesia (pain relief), amnesia (loss of memory),
3

and immobilization.

These agents must be administered intravenously or through inhalation.

There have been many hypotheses over the last century regarding the mechanism(s) of anesthetic
action. It was once believed that the anesthetic entered cells through an unknown way and
caused an unknown disruption in the cell network or that anesthetics interact with regions on the
neuronal cell membrane causing lateral changes in pressure that causes fluidization of the
membrane

4-6

, or even that the anesthetic interacts directly with proteins in the membranes
7

causing analgesia, amnesia, and immobilization .
Acts of crude anesthesiology have been reported since 1846, when patients were given
diethyl ether vapor, ethanol (to the point of intoxication), or opioids before limbs were
1

8

amputated, wounds sewn, or bullets removed from the body.

Prior to the 1840’s, doctors and

dentists would only perform surgical procedures that were absolutely necessary to save an
individual’s life since the patient had to endure pain with no relief because they were conscious
9

during the surgery.

Many patients would die from bacterial infection contracted during or after

surgery, due to both sanitary conditions and the use of surgery as a last resort. In 1846, a dentist,
William Morton

10

, showed that if a cloth was soaked in diethyl ether and held to the mouth and
9

nose of the individual, after some time the individual would appear to be in a sleeping state.

There was, however, no way to control the dosage of inhaled ether fumes and the patient could
either awaken during the surgical procedure or die from an anesthetic overdose. In addition to
dosage issues, the storage and transportation of ether was also dangerous for reasons of
flammability. Subsequent to the discovery of ether as an inhaled anesthetic, other compounds,
including chloroform and nitrous oxide,

11

were discovered. While these compounds were also

potentially dangerous in terms of dosing, they were safer to transport. The dosing of inhaled
anesthetics continues to be problematic, with the attendant issues of monitoring being relegated
to vital sign measurement and observation (e.g. heart rhythm, breathing changes, lower blood
9

pressure, nausea, and vomiting ).
The development of hollow needles (1844)
13

the intravenous (IV) drip (1930),

12

12

led to the creation of syringes (1853) , and
11

allowing the IV introduction of anesthetics.

In 1932, the

first successful barbiturate, thiopental (Figure 1.1), was introduced and was used widely as an IV
14

anesthetic.

Barbiturates have many disadvantages, including their depressant effect(s) on the

2

cardiovascular, the pulmonary, and the neurological systems.

As with ether, a barbiturate

overdose can cause death. Two clinical research groups reported independently the use of
thiopental as an IV anesthetic,

15,16

and this compound is still in use today. Thiopental is

administered in a liquid form directly into the blood stream where it can easily cross the blood
brain barrier (BBB).

Figure 1.1: Thiopental, the first IV anesthetic discovered independently in the 1950’s by two
separate groups.

Current research has shown that general anesthesia produces a combination of
neuropharmacological actions that are specific to each individual neuronal pathway in different
regions of the central nervous system (CNS), in proportion to the dose administered.

11,16

This

means that a particular anesthetic will elicit different responses (or release of different
concentrations of neurotransmitters) from different regions in the brain, these changes are
dependent on the drug administered, the concentration administered, the duration of
administration, as well as the individual themselves. Since the 1930’s, researchers and medical
professionals have determined that a titration of amnesia, analgesia, and immobility drugs over
time is much safer than administering a single large bolus of anesthetic drug at the time of initial

3

sedation. From the onset of anesthetic administration the dose, time, and particular anesthetic
administered was not an exact science because the mechanism of anesthetic action was unclear,
due to the lack of knowledge and instrumentation available to researchers. From 1899 to 1997
there have been four leading hypotheses of anesthetic action: the Meyer – Overton Correlation
6

(1899), the Critical Volume Model of the Lipid Hypothesis of General Anesthetic Action
17

18

(1973) , the Membrane Protein Hypothesis of General Anesthetic Action (1984) , and most
7

currently the Modern Lipid Hypothesis (1997) . The latter two hypotheses are found in the
current literature.

7,19

The Meyer – Overton hypothesis was developed independently by Hans Horst Meyer in
4

5

1899 and by Charles Ernest Overton in 1901 . Each scientist found that general anesthetic
potency was proportional to the solubility of the anesthetic in a hydrophobic solvent, (olive oil)
which was in contact with hydrophilic region (water) causing a water-oil interface.

4-6

This oil-

water interface mimicked the cell wall – cytoplasm interface in the body. Meyer and Overton
determined that this relationship occurred regardless of whether administration was by inhalation
or intravenous. The important quantity was the final concentration of the anesthetic in the lipid
membrane. Pohorille et al. (1998) have concluded that the product of the partition coefficient of
a specified anesthetic dissolved in a nonpolar solvent and its specific anesthetizing partial
pressure (the partial pressure of anesthetic in the blood stream required to induce clinical
6

anestitization) is constant for all anesthetics.

Hence, different anesthetics will produce

anesthesia at different concentration as each other in the lipid bilayer because it is not the
concentration that is important but rather the volume that interacts within the membrane.
4

20

Koblin and colleagues (1990) supported the hypothesis when they reported that 26 different
conventionally inhaled anesthetics that were previously reported to have potencies differing by
as much as six orders of magnitude had the product of the anesthetizing partial pressure and the
21

partition coefficient equal to each other.

Similar results were observed in three different animal
21

models (mice, dogs, and humans) from the same research group.

As studies continued, the

theory that anesthetics penetrate the lipid membrane to access the interior of the cell to cause
anesthesia by interacting with neurons began to lose its acceptance. These doubts were supported
when research had shown that molecules structurally and or chemically similar to the known
anesthetic drugs did not cause anesthetic effects. After 60 years of research, the model of
anesthetic action is split between two lipid solubility theories. These are the Meyer – Overton
hypothesis, where the anesthetic effect is dependent on the molar concentration in a specific cell,
and not on the actual anesthetic itself.

22

The other hypothesis is that the plasma membrane

experiences an increase in fluidity, which is when the hydrophobic regions of the membrane are
caused to expand past the critical volume, upon interactions with anesthetics.

22

Miller and colleagues have theorized that anesthesia occurs when the hydrophobic regions
of the plasma membrane expand beyond a critical volume due to the absorption of inert
anesthetics molecules.

22

The critical volume is defined by the fractional volume of anesthetic to

weight of recipient that is needed to cause full anestitiziation with lack of recovery (EC50
values). They refer to their hypothesis as the critical volume model. While the Meyer – Overton
theory takes into account the molar concentration, the critical volume theory takes into account
the volume fraction solubility in the cell membrane, this means that the researcher is accounting

5

for the amount of the anesthetic in the plasma membrane not what was administered in to the
blood stream.
Anesthetics are a structurally diverse group of organic molecules that include but are not
limited to barbiturates, alcohols, halogenated alkanes, ethers, and some steroids. Most anesthetic
agents are low boiling point liquids that easily volatize at atmospheric pressure and are insoluble
in aqueous solutions. However, some anesthetics, such as barbiturates, are soluble in aqueous
solutions. Researchers have shown that these anesthetics fluidize not only synthetic
phosphatidylcholine – cholesterol lipid bilayers but also some biological membranes; especially
those of the neuron.

23-27

Similar yet larger organic molecules, for instance larger alkanols or

halogenated alkanes, do not fluidize lipid bilayers or biological membranes, and they do not
exhibit anesthetic behavior.

24

Miller and Pang determined that a difference in lipid composition

was a factor that aided in the ability of a molecule to cause an anesthetic effect. This would allow
for a plausible mechanism to be formulated for the depression of neuronal function in individuals
who are in a state of anesthesia.

17

The authors found that lipid bilayers that have 4 – mole %

phosphatidic acid, 33 – mole % cholesterol and the remainder 63 – mole % phosphocholine
mimic the biological nerve membrane. Synthetic membranes of this composition are affected by
anesthetics and fluidize in their presence.

17,24,25

While anesthetics are referred to as non-specific drugs, they must have some
+

+

pharmacological specificity through attraction of membrane processes, such as the Na /K
ATPase pump of red blood cells

26

27

or that of the synaptosome.

These processes, however have

been reported to be unaffected by anesthetics, even at levels that should cause lysing of

6

anesthetic fluidizing cells. The work done by Miller and Pang shows that fluidizing efficacy,
which is the rate of change of membrane fluidity in comparison to the concentration of the
anesthetic administered, is now needed to fully understand the mechanism of the lipid solubility
of anesthetics.

17

Hence, to know the magnitude of fluidizing efficacy or the rate of change of

membrane fluidity with concentration of the anesthetic in the membrane needed to enter the lipid
membrane to cause enough perturbation in order for anesthetic effects to occur can only be
evaluated when the membrane partition coefficient of the particular anesthetic is known.

17

Anesthetics can only act on regions of the membrane that exhibit positive fluidizing efficacies,
this can account for the selectivity of the anesthetics for neuronal cell membranes over the
membranes of red blood cells. However, when looking at biological cells this straightforward
calculation is often complicated by one of two limitations; the first being that there must be
transmembrane proteins in the membrane that can alter the fluidization efficacy of the
28

membrane

or second, that the lipids are distributed heterogeneously across and in the plane of

the membrane.

29

The fact that membrane spanning proteins can affect the fluidization of the

membrane was one of the first indications that membrane protein(s) might play a role in the
action of anesthetics.
While these two hypotheses were operating for over 70 years, they had four common
weaknesses. One issue is that, stereoisomers while having identical partition coefficients into the
30

lipid membrane, exhibit enantiomer-specific anesthetic effects . For example albuterol (2(hydroxymethyl)-4-(1-hydroxy-2-tert-butylamino-ethyl)-phenol) (Figure 1.2 A) contains both
enantiomers and Xopenex® (4-[2-(tert-butylamino)-1-hydroxyethyl]-2-(hydroxymethyl)phenol)

7

(Figure 1.2 B) only contains one, hence Xopenex

®

have far less side effects on the

cardiovascular and pulmonary system than does abuterol.

Figure 1.2: A. Albuterol has 2 stereoisomers one of which causes undesirable side effects B.
®

Xopenex contains the purified R isomer of albuterol.

Another issue not addressed by the plasma membrane hypothesis is the ability of chemically
similar compounds to produce analgesia and amnesia but not immobility. Some compounds
produce convulsive effects indicating that these drugs not only mediate interactions within the
central nervous system but also in the peripheral nervous system.
8

31

There is also a cutoff effect

that needs to be accounted for. The addition of methylene groups to anesthetic and anestheticlike molecules can increase the solubility of these compounds in non-polar media, which in
essence should increase anesthetic effects. Pringle et al. observed that there is a limit to the
effectiveness of this strategy.

32

The plasma membrane hypotheses focus on changes in

membrane fluidity, Franks and Leib have reported that changes in membrane fluidity due to
pharmacologically relevant concentrations of anesthetics are so small that similar changes can be
seen by increasing the environmental temperature of membrane by 1 C.

33

Such changes in

temperature fall within physiologically observed variations. If anesthetics cause an in increase in
fluidity e uivalent to 1 C, the viability of the critical volume hypothesis is called into question.
The mechanism of anesthetic action thus remains open to question and there is credence in the
possibility that membrane bound proteins are involved in mediating aesthetic action.
Franks and Lieb have reported that the mechanism of general anesthetic action is governed
by competitive interactions with endogenous ligands at the active binding sites for specific
receptors.

18

They studied the effects of anesthetics on membrane bound proteins in crystalline

structures completely free of lipids and other cellular structures. In that work the authors studied
the transmembrane protein Luciferase, which, when combined with luciferin in the presence of
adenosine triphosphate (ATP), Mg2+, and O2 is formed with the emission of a photon.

18

Their

data were consistent with a competitive mechanism of action for luciferin and anesthetic at the
active site at the protein,

18

Over the next decade research continued to support the mechanism

that general anesthetics bound directly to transmembrane ligand – gated ion proteins to cause the
anesthetic effect. In a review article, Franks and Lieb concluded that stereospecific general
anesthetic isomers had an effect on ligand-gated ion channels such on the glutamate receptor, the
9

nicotinic acetylcholine receptor, and γ–aminobutyric acid A (GABAA) receptor families.

19

Support for this mode of action for anesthetics binding to proteins is found in the mechanism of
operation for other pharmaceuticals. A host of drugs bind directly to target proteins to produce
the desired effect as well as some other undesirable effects.

19

The modern lipid hypothesis proposed by Cantor considers lateral pressure profiles in
7

membranes. Most likely anesthetics interact with transmembrane proteins causing an inhibition
of the ligand – gated ion channel in proteins.

7

In this model, Cantor discusses that if the ion

channel does not open due to the binding of the anesthetic then the area of the membrane where
the ion channel should open will increase in cross – sectional area closer to the aqueous interface
than in the interior of the membrane causing a change in the lateral pressure of the membrane
since their measured change in cross sectional area is greater that the aqueous lipid interface than
7

at the center of the lipids where the acyl chains interact. This hypothesis is supported by lateral
pressure profiles constructed via thermodynamic analysis of synthetic lipid bilayers. While
Cantor’s research provides a plausible hypothesis, he neglected to add proteins to his bilayers or
use real cellular membranes and he still referenced that the anesthetics most likely bind to the
proteins; therefore, the leading hypothesis of anesthetic action is that they interact with ligand –
gated ion channel proteins.
Ligand – gated ion channels are the simplest membrane – bound protein channels that open
or close upon the binding of the specific ligand. These ion channels mediate the flow of ions
across the membrane through either a specific or nonspecific ion pore. The flow of ions across a
membrane causes secondary messaging systems to operate leading to multiple conscious and
unconscious mediations of physical responses from breathing, memory, and heart rate to name a
10

few. Therefore, the ability to cause known modulations in macroscopic responses like the
depression of memory, pain, and movement with anesthetics is advantageous to the medical and
research communities for surgeries, but the microscopic cellular level operation of these
compounds remains to be understood. It is important to determine if anesthetics cause
perturbations in the lipid polar head group region of membranes, if the perturbations occur in the
non-polar acyl chains region, or if the interactions between anesthetics and plasma membranes
do not involve the bilayer structure directly.
Recent work has suggested anesthetic interactions with proteins in the cell membrane with
either animal model or cell lines. Researchers investigating anesthetic interactions with
transmembrane proteins are doing so in vivo, in vitro, and ex vivo. The model organism is either
the Sprauge Dawley or Wistar rat, with the cortex or hippocampus regions used for experiments.
These regions were chosen because they are rich in the ligand – gate GABAA receptors. Hirota et
al. reported that, using square waveform electrophysiology measurements on harvested rat
hippocampus that were transversely sliced at thicknesses 400 nm, an enhanced release of the
ready reserve pools of GABA vesicles from the presynaptic terminal was seen upon treatment
with intravenous (IV) anesthetics.

34

The anesthetics used were sevoflurane (Figure 1.3 A),

isoflurane (Figure 1.3 B), thiopental (Figure 1.3 C), pentobarbital (Figure 1.3 D), and nipecotic
(Figure 1.3 E); the first two are inhalation anesthetics and the latter 3 are IV anesthetics.

11

34

Figure 1.3: A. Sevoflurane; B. Isoflurane; C. Thiopental; D. Pentobarbital; E. Nipecotic.
12

These findings may explain why individuals who are anesthetized frequently due to multiple
surgeries from trauma are more often unsuccessfully immobilized. GABA is the principal
inhibitor in the CNS and, when there is a larger release of GABA in the synaptic cleft due to the
action of anesthetics, the clearing or decay time of GABA from the cleft is slowed, allowing a
greater response to GABA. The Akaike group investigated the effects of volatile anesthetics on
isolated GABA synapsis from Wistar rats using whole cell patch clamp electrophysiological
techniques.

35

They harvested the hippocampus of rats, which was kept in ice cold oxygenated

(O2) artificial cerebral-spinal fluid (aCSF) until 400 nm thick sections were taken for patch
clamp measurements.

35

Sevoflurane (Figure 1.3 A), isoflurane (Figure 1.3 B), and enflurane

(Figure 1.4) all enhanced the exogenous levels of GABA upon stimulation.

35

Figure 1.4: Structure of Enfurane.

Anesthetics cause an increase in chlorine ions to pass into the neuron through the GABAA
receptor which causes the GABAA receptor to become inhibited, but the mechanism by which
this occurs is unknown. Turina posed a plausible explanation with the use of the drug propofol.
Propofol (2, 6-diisopropylphenol) (Figure 1.5) causes a narrowing of the neuritis by causing an
increase in the contractibility of myosin of the GABAA receptor.
13

Figure 1.5: Propofol is a commonly used barbiturate anesthetic for both animals and humans.

This narrowing causes a measurable response of the GABA-filled vesicles that were
reported to have a change in direction and increase in velocity of neurotransmitter release from
the docked vesicle towards the cell body instead of into the synaptic cleft. These data were
collected using cortex tissue harvested from newborn Sprague Dawley rats. The harvested tissue
was kept alive with warmed O2 – artificial cerebral spinal fluid (aCSF) while being monitored
with video microscopy.

36

The narrowing of the neuritis via the increase in the contractibility of

myosin of the GABAA receptor was observed only on the harvested cortex tissue but when the
GABAA receptor antagonist gabazine showed a reversal of the effects of the propofol. The
authors state that their work is a step in the direction of understanding the mechanism of
anesthetic action but further investigation is needed.

36

These are initial steps in determining the

mechanism of anesthetic action. A broad selection of anesthetics will need to be investigated
before commonality in two models of action can be resolved. This is further supported by the
fact that GABA receptor itself has four subunits of interaction are the α1, α2,  and β1 subunits.
The ribbon structure of the GABAA receptor is shown in Figure 1.6.

14

Figure 1.6: Refined structure of the GABAA receptor at 4A resolution. Unwin, N., (2005)
J.Mol.Biol.346: 967. For interpretation of the references in color in this and all figures, the
reader is referred to the electronic version of this thesis.

Despite this evidence, the role of anesthetic interactions directly with lipid bilayers cannot
be ruled out. Nelson et al. (2012) investigated lateral pressure changes in the head and tail
regions of DPPC liposomes when dosed with isoflurane with two fluorescent chromophore using

15

steady state fluorescence anisotropy measurements.

37

Anisotropy decay measurements is an

experimental result that gives rise to how fast a chromophore orientates and diffuses out the local
environment in the Cartesian component axis of X, Y, and Z. Vesicles were formed using
extrusion with 1,6-diphenyl-1,3,5-hexatriene (DPH) (Figure 1.7 A) or N,N,N–Trimethyl–4-(6–
phenyl–1,3,5–hexatrien –1– yl) phenylammonium p–toluenesulfonate (TMA – DPH) (Figure 1.7
B) as probes and 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine (DPPC) as the phospholipid
(Figure 1.8) in an isoflurane solution prepared in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid (HEPES) buffer.

Figure 1.7: A. DPH is a non-polar chromophore; B. TMA-DPH is polar
16

Figure 1.8: Structure of DPPC

The Nelson group reported an increase of 0.2 in the steady state fluorescence anisotropy
value for TMA – DPH vesicles and a similar decrease in the steady state fluorescence anisotropy
value of DPH-containing vesicles when the vesicles were treated in 7 – 25 mM isoflurane baths.
37

The authors attribute the increase in non - axial dye mobility in the tail region and the

decrease in the head region to the lateral pressure changes in the membrane associated with the
interaction of the anesthetic.

37

While this information has provided some insight into the
17

mechanism of general anesthetic action, much remains to be done. A key step in understanding
anesthetic action is to resolve whether or not the primary chemical interactions are between
anesthetic and the lipid bilayer or between anesthetic and transmembrane proteins. We are poised
to evaluate whether or not anesthetics interact with lipid bilayers using time resolved
fluorescence lifetime and anisotropy decay measurements.

These measurements provide

significantly more molecular-scale insight into probe-lipid interactions than can be obtained with
steady state fluorescence anisotropy measurements.
The goal of this research is to investigate the interaction of general anesthetics with
synthetic lipid membrane and to identify changes in the organization of the lipid membrane. The
Modern Lipid Hypothesis was investigated with the use of synthetic vesicles. The Membrane
Protein Hypothesis of General Anesthetics was not investigated here due to experimental
complexity. Lipid bilayers in the form of vesicles were made to mimic the neuronal cell plasma
membrane. The vesicles were unilamellar of a controlled size of 400 nm. These vesicles were
constructed with one of two probes so that either the polar head regions of the lipids or the nonpolar acyl tail regions were interrogated by rotational diffusion of the probes.
Synthetic vesicles are produced by extrusion, as described previously.

38

The composition

of 63 – mole % phosphatidylcholine (PC), 4 – mole % phosphatidic acid (PA) with 33 – mole %
cholesterol is used in the synthetic vesicles studied in this work. The PC that will be used to
make the synthetic vesicles is 1, 2-dihexadecanoyl-sn-glycero-3-phosphocholine (DPPC) (Figure
1.8).

Two different chromophores will be used to investigate anesthetic identity- and

concentration-dependent changes in the vesicle head group and acyl chain regions. These are
(N-(7–nitro–2-1,3–

benzoxadiazol–4-yl)

(NBD),)

1,2–dioleoyl–sn–glycerol–3–

phosphoethanolamine N-(7–nitro–2-1,3– benzoxadiazol–4-yl) (ammonium salt) (Figure 1.9),
18

and perlyene (Figure 1.10). The phosphatidic acid (PA) used is 1,2–dihexadecanoyl–sn–
glycerol–3-phosphate (sodium salt) (DPPA) (Figure 1.9). Cholesterol (Figure 1.11) was obtained
from ovine wool. These vesicle constituents were chosen to model qualitatively the composition
39

of neuronal cell plasma membranes based on the work of Botchway et al.

19

Figure 1.9: Left DPPE with an NBD chromophore tag, 1,2-dioleoyl-sn-glycero-3phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (ammonium salt). Right DPPA, 1,2dihexadecanoyl-sn-glycero-3-phosphate (sodium salt).

20

Figure 1.10: Structure of Perylene.

Figure 1.11: Structure of Cholesterol.

Fluorescence is a radiative emission process that occurs as a result of the excitation of a
sample by absorption of a photon. Optical absorption is described using Beer’s Law,
(1.1)
21

-1

wehere A is the absorbance, ε is the molar absorptivity (L (mol cm) ), b is the path length of the
-1

cell (cm) and c is the concentration of the absorbing species (mol L ). Upon excitation,
radiative emission can occur as either fluorescence (short lifetime; < 10
phosphorescence (long lifetime; seconds to minutes).

-5

s) or as

The difference between these two

emission processes is that with fluorescence the electronic energy transitions do not involve a
change in electron spin, hence the shorter lifetime. This emission of radiation as a photon occurs
at wavelength longer than that of the excitation photon.
Factors that can affect fluorescence intensity are transition type, chromophore structure
and rigidity, system temperature, pH, concentration, and dynamic quenching.

40

Fluorescence

quantum efficiency is the ratio of the radiative rate constant to the sum of all rate constants for
relaxation from the excited state. The rate constants that can contribute include the radiative rate
constant, (kr), intersystem crossing (kin), external conversion (kec), internal conversion (kic),
predissociation (kpd), and dissociation (kd).

40

The individual rate constants are related to

fluorescence quantum yield according to equation 1.2.

(1.2)

The magnitude of each rate constant depends on the chemical structure of the chromophore and
the environment.

Molecules that have aromatic functional groups, aliphatic and alicyclic

carbonyl structures or those molecules that have highly conjugated double bonding character are
typically characterized by a dominant π → π* transition and in many instances they are observed

22

to emit.

40

Factors that can reduce fluorescence quantum yield include the presence of halogen

substituents on ring structures. In relation to structure, planar, rigid chromophores often exhibit
a high fluorescence quantum yield because of the optimization conjugation and minimization of
40

access to nonradiative decay channels.

For such chromophores, fluorescence intensity is

typically temperature dependent. Increased temperature provides increased system energy that
will reduce the viscosity of the solution and increase the frequency of collisional interactions that
can result in energy transfer or internal conversion. Compounds that have acidic or basic ring
substituents can exhibit pH-dependent fluorescence intensity because protonation/deprotonation
will alter the nature of -conjugation in such systems.
As indicated above, for a molecule to fluoresce it must first absorb a photon and since
absorption is related to concentration then fluorescence is also dependent on concentration.
Beer’s law can also be expressed as

40

(1.3)

where P is the power of the exciting light beam after passing through the cell of path length b,
and P0 is the incident power of the exciting light beam. Fluorescence can be related to equation
1.3 through

40

(

)

(1.4)

where F is the fluorescence intensity, K’ is a proportionality constant to account for geometric
and electronic details of the particular instrument being used, K’ in fact, is instrument dependent
23

and the uantum efficiency, and εbc is the absorbance. Equation 1.4 is valid over a limited
concentration range.

Deviations from the relationship between fluorescence intensity and

chromophore concentration can arise due to collisional quenching or aggregate formation, for
example.

These factors need to be taken into account when choosing probes and probe

concentrations for fluorescence spectroscopy measurements.
NBD has a fluorescence lifetime that is sensitive to the dielectric properties of the local
environment this means that in a polar environment it will have a shorter fluorescence lifetime
than in a non- polar environment. The anesthetics in question are characterized by a dielectric
response that is different than either an aqueous environment or a nonpolar environment. When
anesthetics interact with lipids or cholesterol, the dielectric response of the modified
environment will be changed and if this change occurs in close proximity to the NBD
chromophore, a change in fluorescence lifetime and also possibly reorientation time will be seen.
Any change in chromophore reorientation time was observed for a nitroxide reporter group
tethered to the PC by Miller and Pang for range of anesthetics that include but are not inclusive
to pentobarbital and halothane in synthetic lipid membranes. The NBD chromophore is excited at
450 nm and has an emission maximum at 530 nm.

17

For the interrogation of the non-polar acyl

chain region of our vesicles, we use perylene as a probe molecule. Studies from Lapinski and
Blanchard showed that in vesicles ranging from 100 – 1000 nm the chromophore perylene
orientated itself in the acily chain regions of unilamellar vesicles.

41

Perylene was chosen

because it is a non-polar, rigid polycyclic hydrocarbon that does not exhibit anomalous
spectroscopic behavior such as strong vibronic coupling or intensity borrowing,42 and has a
fluorescence lifetime that is well matched to the rotational diffusion times measured in this work.

24

Perylene is excited at 444 nm exhibits prominent emission in the vicinity of 470 nm. When the
saturated PC and PA lipids are in an ordered phase and perylene is located within this region,
relatively slow rotational diffusion motion is measured. Interactions between anesthetics and the
acyl chain region of the lipid bilayer will serve to decrease the organization of the acyl chains,
providing more rational freedom for perylene, resulting in faster reorientation times.
To gain some measure of insight into the effects of anesthetics on the lipid membrane, three
anesthetics have been chosen for examination: barbiturates, ethers and halogenated alkanes. The
specific

anesthetics

are

pentobarbital

pyrimidinetrione) (Figure 1.3 D),

43,44

(5-Ethyl-5-(1-methylbutyl)-2,4,6(1H,3H,5H)-

isoflurane (1-chloro-2,2,2-trifluoroethyl difluoromethyl

ether) (Figure 1.3 B) and halothane (1-bromo-1-chloro-2,2,2-trifluoro-ethanane) (Fig. 1.10).

44

Information on the properties of these anesthetics, including molecular weight, boiling point, and
the Oswald solubility coefficients can be found in Table 1. While the modes of activity of the
anesthetics are thought to be different, there remains much detail to be determined as to the
molecular interactions that characterize the anesthetic-lipid system. The impact of this work will
show whether anesthetics interact with the plasma membrane without transmembrane protein
and if they do where interactions are greatest: in the polar or non-polar regions on the bilayer.
Also, this work will be a stepping stone to help bridge the Modern Lipid Hypothesis with that of
the Membrane lipid Hypothesis of General Anesthetics.
As one can conclude there are significant risks when undergoing anesthesia. Even the
most skilled and trained anesthesiologist may have problems with a patient that is considered
healthy since the mode(s) of action of these drugs remains to be understood in detail. Their
effects are not only on the CNS but also in the peripheral nervous system, with effects on the
heart, lungs, and gastrointestinal tract. It is important to elucidate the interactions between
25

anesthetics and model lipid bilayers to advance our knowledge of the mode(s) of operation of
anesthetics.

Figure 1.12: Structure of Halothane.
Name of
Inhaled
Anesthetic

MW
(Da)

Halothane
Isoflurane

197
184.5

BP
(degree
C) at 1
atm
50.2
48.5

Ostwald Solubility Coefficients

SVP at
20 C
(kPa)

MAC
(% v/v)

O/W

Bl/G

W/G

32.5
31.9

0.75
1.15

220
174

2.3
1.43

0.8
0.62

Br/G O/G
4.8
21

Br/Bl

224
91

1.9
1.6

Table 1.1: Summary of properties of the inhaled anesthetics halothane and isoflurane. MW molecular weight, BP - boiling point, SVP - saturated vapor pressure, MAC - minimal alveolar
concentration, O/W - oil water, Bl/G - blood gas, W/G - water gas, Br/G - brain gas, O/G - oil
gas, Br/Bl brain blood interfaces

44

26

CHAPTER 2
EXPERIMENTAL PROCEDURES
Current investigations on the mechanism of anesthetic interactions with the central
nervous system (CNS) have taken two approaches. One group of investigations utilizes cell lines
and/or animal models, and another body of work uses synthetically prepared vesicles. Groups
who use cell lines and/or animal models do so, on the premise that anesthetics interact with the
ligand – gated ion channels (LGIC) imbedded in the lipid membrane. Upon interaction of the
anesthetic with the LGIC, there are changes in the membrane and with neurotransmitter (NT)
release. There is also another school of thought, based on the range of chemical structures that
give rise to outwardly similar anesthetic effects. For this reason, the processes of primary
concern are the interactions between the lipid membrane and the anesthetics, with such
interactions giving rise to changes in lipid membrane order or permeability, which lead to
changes in NT release. A connection can be made between these two possibilities. We use
synthetically prepared vesicles that mimic the neuronal lipid membrane composition without
having the complexity of LGIC structures in them. With the use of these model bilayer systems
we can investigate the fluidity and extent of organization of the bilayer as a function of exposure
to different anesthetics.

We measure the fluorescence lifetime and induced orientation

anisotropy decay of a chromophore in the membrane using time – correlated single – photon
counting (TCSPC). These experiments are designed to determine the details of interactions
between anesthetics and the lipid bilayer membrane. If anesthetics interact directly with the lipid
membrane, there will be changes in the organization and local inter-molecular frictional
interactions, and these changes will be reflected in the rotational diffusion dynamics of the probe

27

residing in the lipid bilayer acyl chain region. We provide detailed information in this Chapter
on the experimental details of these measurements.
Vesicle Preparation: Vesicles were formed by extrusion from mixtures containing
phospholipid, sterol and chromophore species in predetermined amounts. The formation of
vesicles from mixtures of these components is driven entropically to produce structures where
interactions between polar solution and nonpolar organic constituents are minimized.

The

vesicle constituents 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine (DPPC), 1,2–dioleoyl–sn–
glycerol–3–phosphoethanolamine N-(7–nitro–2-1,3– benzoxadiazol–4-yl) (ammonium salt)
(18:1 NBD-PC), 1,2–dihexadecanoyl–sn–glycerol–3-phosphate (sodium salt) (DPPA), and
cholesterol were purchased from Avanti Polar Lipids Inc. (Alabaster, AL) in powdered form and
were used without further purification. Perylene was purchased from Sigma-Aldrich (St. Louis,
MO) and used as received. These constituents were dissolved in 10 mM 2-Amino-2hydroxymethyl-propane-1,3-diol (TRIS®-HCl) – buffer containing 100 mM sodium chloride
( igure .1 at p

.4, they were then stored at - 0 C until use. Both

B

and perylene

chromophore-containing vesicle solutions were prepared in 0.3 mM concentrations where 63
mole-% was DPPC, 33 mole-% cholesterol and 4 mole-% DPPA. Vesicles containing 18:1
NBD-PC chromophore had a chromophore concentration of 1 mole-% and vesicles containing
perylene had a chromophore concentration of 0.5 mole-%. The TRIS®-HCl buffer was prepared
using Milli-Q Plus water purification system (Millipore, Bedford, MA). To ensure adequate
mixing, the lipid mixtures underwent five free e-thaw-vortex cycles. ach cycle was a 5 minute
immersion in li uid nitrogen, followed by a 5 minute immersion in a 0 C water bath, and a
minute vortex. To prepare 400 nm diameter vesicles, these mixtures were extruded eleven times
through a polycarbonate membrane with a 400 nm pore diameter (Avanti Polar Lipids Inc.,

28

Alabaster, AL) in 1 mL increments. It is known from prior work in the Blanchard group that this
method of vesicle preparation produces vesicles with the same diameter as the nominal pore size
of the membrane.41 All extrusions were performed at room temperature. A 20 mL aliquot of
lipid mixture was used to prepare vesicles by extrusion weekly, and these vesicles were stored at
room temperature until used within the same week.

Figure 2.1: Structure of Tris - HCl ®.

Anesthetic Solution Preparation:

Pentobarbital,

(5-Ethyl-5-(1-methylbutyl)-

2,4,6(1H,3H,5H)-pyrimidinetrione), (Figure 1.3 D) was donated by Professor Spence and was
purchased originally from Sigma-Aldrich (St. Louis, MO). Isoflurane, (1-chloro-2,2,2trifluoroethyl difluoromethyl ether), (Figure 1.3 B) was purchased from Phoenix Pharmaceutical
Inc. (St. Joseph, MO), and halothane, (1-bromo-1-chloro-2,2,2-trifluoro-ethanane), (Figure 1.12)
was purchased from Sigma-Aldrich (St. Louis, MO). All anesthetic solutions were prepared
weekly in 10 mM or 100 mM stock solutions in Milli-Q water (Millipore, Bedford, MA). These
solutions were stored in tightly sealed amber bottles in a dark room.
Steady-State Fluorescence Measurements: Excitation

and

emission

spectra

were

collected using a Hitachi F-4500 fluorescence spectrophotometer to determine the NBD (Figure
1.7) and perylene (Figure 1.8) absorption and emission band positions in our vesicle

29

preparations. A spectral band pass of 1 nm was used for both the excitation and emission
monochromators for all measurements.
Time-Correlated Single Photon Counting (TCSPC):

A

time-correlated,

single-photon

counting instrument was used to acquire time-resolved fluorescence lifetime and fluorescence
anisotropy decay data. The details of this system have been described previously,

45-47

and only

the essential properties will be recapped here. The light source is a CW passively mode-locked,
diode-pumped Nd:YVO4 laser (Spectra Physics Vanguard) that produces ~12 ps pulses at 1064
nm at a repetition rate of 80 MHz. The fundamental wavelength is frequency doubled to produce
2.5 W average power at 532 nm with the same pulse characteristics as the fundamental output.
Pulses at 355 nm were generated by mixing the fundamental and second harmonic pulses,
producing 2.5 W average power with the same pulse characteristics as with the fundamental and
second harmonic output. The second and third harmonic outputs from the Nd:YVO4 laser excite
cavity dumped dye lasers (Coherent 702-2) that operate between 430 – 850 nm. The cavity
dumped dye laser produces a 5 ps pulse at a repetition rate of 4 MHz. Average output power of
the dye laser is wavelength dependent and is typically in the range of 50 mW to 250 mW.
Excitation of the NBD S1 ← S0 transition was at 450 nm (Stilbene 420 dye, Exciton), and
emission was collected at 530 nm. For perylene, excitation of the S1 ← S0 transition was at 440
nm (Stilbene 420 dye, Exciton), and emission was collected at 475 nm. The laser output was
linearly polarized with a polarization extinction ratio of ca. 100:1. Each polarized excitation
pulse is divided into two pulses, with one pulse directed to a reference photodiode (Becker &
Hickl PHD-400-N) and the other pulse directed into the sample. Emission from the sample is
collected at right angles to the excitation axis using a 40 X reflecting microscope objective
(Ealing) and directed to two detection channels. The emission signal is separated into two
30

polarization components using a polarizing cube beam splitter, parallel (0
( 0

and perpendicular

with respect to the vertically polari ed excitation pulse. The separate polarized signal

components are detected simultaneously using microchannel plate photomultiplier tubes (MCPPMT, Hamamatsu R3809U-50). Each photomultiplier tube is equipped with a subtractive double
monochromator (Spectral Products CM-112). The parallel and perpendicular transients are
recorded separately by two – channel TCSPC detection electronics (Becker & Hickl SPC-132)
yielding ca. 30 ps response functions for each detection channel.

All instrumentation was

operated using a LabVIEW® (National Instruments) program written in-house. All samples were
mixed by inverting 4 times between data acquisitions to minimize photo-bleaching effects. A
schematic of the TCSPC instrument is shown in Figure 2.2.

Figure 2.2: Schematic of TCSPC instrument used in this work.

31

Fluorescence Lifetime and Anisotropy decay:

luorescence lifetime (τfl) is the time

constant associated with the first-order population decay from the first excited singlet state of a
molecule. The rate constant for the relaxation process is given by k:
(2.1)
where kr is the rate constant of the radiative decay and knr is the rate constant of the nonradiative decay. Because the intensity of fluorescence is related directly to the population of the
excited state, the time-dependence of the intensity decay is modeled as a first order process. The
time constant of the decay is inversely related to the rate constant:
( )

⁄

(2.2)

The time-dependence of first order relaxation is given by equation (2.3)
( )

( )

(

( ))

(2.3)

where t is time, I0 is the initial intensity at time zero, and τfl is the fluorescence lifetime. The
fluorescence lifetime is determined by fitting the experimental time domain fluorescence
intensity decay to equation 2.3. The excited and emitting transitions can be described with
respect to the Cartesian axis system for the chromophore.

Molecules absorb light most

effectively when the polarization of the exciting electric field is parallel to the orientation of the
absorbing transition dipole moment, with the relative efficiency of this process scaling according
to Equation 2.4,
( )

(

)

(2.4)

where θ is the angle between the absorbing transition moment and the polari ation of the incident
electric field.

32

In the true domain there are two contributions to the intensity decay of polarized
transients. These are the population of excited molecules and their orientation distribution. To
separate the contributions of these two processes, the emission intensity decay can be measured
at a polarization angle where orientation relaxation does not contribute. For linear processes the
angular dependence of emission polarization is given by Equation 2.4, and the magic angle is
given by the condition (θ

0. or this condition θ 54. .

To extract information from polarized emission transitions on the molecular motion of
the excited ensemble, the induced orientation anisotropy function is used
R=

( )

( )

( )

( )

(2.5)

The anisotropy decay function is the time resolved difference between polarized emission
transients, normalized for total fluorescence intensity as a function of time. Physically this
quantity represents the time scale and means by which a non-random distribution re-randomizes.
The functional form of R(t) has been treated theoretically and can contain up to five exponential
decay components. Experimentally, one or two component decay can be resolved, with the time
constants and relative amplitudes of the components provide information on the physical
properties of local environment of the chromophore and the shape of the volume it sweeps out.
The key to interpreting R(t) data lies in extracting the Cartesian components of the rotational
diffusion constant from the exponential decays. This issue is discussed in detail for perylene in
Chapter 3.
The simplest model for rotational diffusion was developed in 1929 by Debye, and
subsequently modified by Perrin to account for rotor shape, and Hu and Zwanzig to account for
frictional interactions between the rotor and its environment. This model indicates that the time
constant of the induced orientation anisotropy decay depends on the viscosity of the medium (η ,
33

the shape of the rotating entity (S) (for perylene S = 0.7), and the hydrodynamic volume of the
rotor (V) (V = 225 Å3 for perylene).
(2.6)
where τor is the anisotropy decay time constant, Drot is the rotational diffusion constant, f is a
term to account for frictional

component of the interactions between the rotor and its

environment, kB is the Boltzmann constant and T is the system temperature.
The time-evolution of the experimental R(t) signal is schematized in Figure 2.2 and in
some instances the modified DSE equation (Eq. 2.6) can be applied directly. For other systems,
including perylene reorienting in vesicle structures, extracting information on the details of
molecular rotational motion is somewhat more involved than the simple application of Eq. 2.6
and we discuss that issue in Chapter 3.

Time

0
Time

Figure 2.3: Schematic of the principles behind the anisotropy decay measurement.

34

CHAPTER 3
The central purpose of this work is to understand the manner in which general anesthetics
interact with model lipid bilayer structures. There have been a number of mechanisms proposed
for the operation of general anesthetics, among them being the hypothesis that general anesthetic
molecules interact with lipid bilayer structures in such a way as to disrupt the organization of the
bilayer and thus alter the ability of the bilayer to mediate transport processes. It is not clear
whether this transport mediation acts through the structure of transmembrane proteins or directly
on the bilayer, but the common underlying premise is that the anesthetic molecules interact with
the lipid bilayer.
There are a number of means available to characterize the organization of lipid bilayer
structures. Among them is the use of fluorescent chromophores imbedded in the bilayer or
attached to a bilayer constituent. While fluorescent chromophores can produce local structural
perturbations due to their size and/or polarity, their use is well established and much has been
learned from their use regarding lipid bilayer structure and dynamics. In this work we have used
two fluorescent probes, one attached to the head group of a phospholipid and the other free to
partition into the nonpolar region of the bilayer. We have used a phosphoethanolamine-N-(7nitro-2-1,3-benzoxadiazol-4-yl) (ammonium salt), (NBD) and perylene (Fig. 3.1). The tethered
NBD species locates the chromophore in the lipid bilayer polar head group region and perylene,
which is not attached to a lipid bilayer constituent, exhibits essentially no solubility in water and
partitions exclusively into the bilayer acyl chain region. Acquisition of time-resolved

35

perylene
Excitation
1.0 1.0
Emission
emission

excitation

Normalized Intensity

normalized intensity

0.8 0.8
0.6 0.6
0.4 0.4
0.2 0.2
0.0 0.0

300

300

350

350

400

400

450

450

500

500

550

550

600

600

wavelength (nm)

Wavelength (nm)

Figure 3.1. Left: Structure, excitation and emission spectra of perylene

fluorescence data from the tethered NBD chromophore as a function of the amount of anesthetic
present has shown that this chromophore is not sensitive to the presence of anesthetics. This is
not a surprising result owing to the polarity of the region in which the NBD chromophore
resides.

Perylene, which incorporates exclusively into the lipid bilayer acyl chain region,

exhibits rotational diffusion dynamics that depend significantly on the concentration of
anesthetic present in the system, and it is the results for this chromophore which are the focus of
this Chapter.
Perylene has been studied in lipid bilayer structures before.

41,48-51

Measurements on

perylene in DMPC bilayers as a function of system temperature revealed the sensitivity of this
36

chromophore to phase transitions within the lipid acyl chain region.

41,52

DMPC exhibits a gel-

to-fluid phase transition at 24C and, for perylene incorporated in DMPC vesicles less than ca.
800 nm diameter, this phase transition influences the reorientation dynamics significantly.

41

For vesicles of 1 µm diameter and larger, the reorientation dynamics of perylene are no longer
sensitive to the phase transition.

41

This is because for small diameter vesicles the structural

asymmetry between the inner and outer leaflets is sufficient to cause perylene to partition
preferentially into one of the leaflets.

41

For larger diameter vesicles the structural asymmetry

between the inner and outer leaflets becomes negligible and the chromophore resides in the interleaflet gallery.

41

For the work described here, perylene is used as the chromophore because of

its ability to sense local organization and the vesicles are maintained at a diameter of ca. 400 nm
in order that the chromophore will reside within one of the lipid leaflets rather than in the interleaflet gallery.

41

Two bodies of data have been measured for perylene in vesicles composed of
phospholipid, sphingolipid and cholesterol designed as a simple model of the composition of
neuronal cell plasma membranes.

These bodies of data are the fluorescence lifetime and

fluorescence anisotropy decay of perylene in vesicles as a function of exposure to varying
concentrations of three anesthetics added to the solution. The anesthetics used are pentobarbital
(Figure 1.3 D), halothane (Figure 1.10) and isoflurane (Figure 1.3 B). These general anesthetics
were chosen because they are representative examples with structures that differ significantly
from one another. Fluorescence lifetime data for perylene may serve as an indicator of local
environment, but there is no fundamental theoretical connection between the fluorescence

37

lifetime behavior of a comparatively large organic chromophore and its response to changes in
local environment. For this reason, fluorescence lifetime data can be informative at a qualitative
level, at best, for the systems reported here.
Fluorescence anisotropy decay data, however, has a well-established theoretical
framework within which they can be interpreted.

53-56

It is information from these data that are

most useful in revealing the effects of general anesthetics on lipid bilayer structures. Before
considering the experimental data in detail, the theoretical basis for their interpretation is
presented. The operative mechanism for fluorescence anisotropy decay in the systems examined
here is the rotational diffusion of the chromophore. In solution, there exists an orientationally
random distribution of chromophores prior to optical excitation. Excitation of the sample with a
vertically polarized pulse of light photo selects an orientationally non-random subset of all
chromophores present. This nonrandom subset of chromophores re-randomizes after excitation
and it is the rate(s) and manner in which this relaxation process occurs that provides insight into
local restrictions on the ability of the chromophore to rotate. Such restrictions on chromophore
motion reveal local organization in the system and characterizing them as a function of changes
in the system (e.g. anesthetic concentration) provides insight into the interactions between
anesthetic and lipid bilayer acyl chains.
For fluorescence anisotropy decay measurements, polarized emission transients I||(t) and
I(t) are combined to produce the induced orientation anisotropy function, R(t) (equation 2.5). It
is the functional form of R(t) that contains chemical information. Chuang and Eisenthal have
developed the theoretical basis for relating R(t) to the rotational diffusion constant of the
chromophore, D =

⅓(Dx+Dy+Dz), and the relative orientations of the absorbing and emitting
38

transition dipole moments.

53

To apply the Chuang and Eisenthal theory, the Cartesian axes of

the chromophore are assigned, with the axis of the absorbing transition dipole moment defining
the x-axis.

53

For perylene, the S1 ↔ S0 transition dipole moment is oriented along the

molecular long axis in the plane of the  system. For the discussion that follows, the y-axis for
perylene is the short in-plane axis and z is perpendicular to the chromophore  system plane.
For a spherical rotor, where the Cartesian components of the rotational diffusion constant
are all equal, the anisotropy decay time is related directly to D through the Debye-StokesEinstein equation

 OR  6D 1 

54-56

,

V
(3.1)

k BT

Where η is the viscosity of the surrounding medium, V is the volume of the chromophore (for
perylene V = 225 Å3)

57

, kB is the Boltzmann constant the T is the temperature. It is the rare

exception for chromophores to sweep out a spherically symmetric volume as they rotate. In
situations where the Cartesian components of D, are unequal, two model cases can provide
insight into the motional properties of the chromophore. Specifically, when Dx>Dy=Dz, the
chromophore is termed a prolate rotor, and for Dz>Dx=Dy the chromophore is termed an oblate
rotor.

rom Chuang and isenthal’s work,

53

for x-polarized absorbing and emitting transitions,

a prolate rotor (Figure 3.2 C) will decay as

R(t )  0.4exp( 6Dz t )

(3.2)

and an oblate rotor (Figure 3.2 B) will decay as

R(t )  0.1exp((2Dx  4Dz )t  0.3exp( 6Dxt )
39

(3.3)

A

B

X

Z

Z

Y
X
Y

Figure 3.2: A. How perylene would orientate in the Cartesian components if it were a prolate
rotor B. How perylene would orientate in the Cartesian components if it were an oblate rotor
Thus, the number of exponential decays contained in R(t) is determined by the asymmetric
nature of the chromophore rotational motion. For the measurements reported in this work, twocomponent anisotropy decay is seen in all cases. From the experimental data and the use of
Equation 3.4, it is possible to extract Dx and Dz, and from the definition of an oblate rotor (vide
infra), Dx = Dy, allowing D to be determined.
Experimentally, two time constants are recovered from the anisotropy decay data. These time
constants are related to Dx and Dz according to Equation 3.4 and 3.5

53

,

(3.4)

40

(

)

(3.5)

The quantities of interest are the values of Dx (=Dy) and Dz, and the ratio Dz/Dx as a function of
anesthetic concentration. We present these data in Tables 3.1 and 3.2, and in Figures 3.3 through
3.6. The time constants in Table 3.1 were extracted from experimental data. What can be
observed from the data tabulated data in 3.1 is that there is not a dose dependenty trend in the
data set. For the fluorescence lifetime measurements the data were fitted to the function

ffl(t)=I0exp(-t/fl) and for the anisotropy decay measurements the data were fitted to the
function fR(t)=R1(0)exp(-t/OR1) + R2(0)exp(-t/OR2).

41,45,46,53

Since there is no obvious

does dependent trend in the data it is vital to break the data up into the Cartesian components of
Dx, Dz, and Dz. In Table 3.2, the quantities Dx, Dz, Dz/Dx, DSE and ηDSE were derived from the
experimental time constants shown in Table 3.1 and Equations 3.1 and 3.3 – 3.5.

41

Control
Pentobarbital

Isoflurane

Halothane

Concentration
(mM)
0
1
2
5
7
10
2
3
5
7
10
2
3
5
7
10

fl (ps)

OR1 (ps)

OR2 (ps)

6904 ± 14
6705 ± 18
6741 ± 19
7011 ± 20
6672 ± 17
6960 ± 17
6913 ± 17
6967 ± 19
7380 ± 21
7159 ± 18
6872 ± 18
5977 ± 9
5241 ± 10
7128 ± 21
7178 ± 21
5112 ± 16

225 ± 93
63 ± 30
40 ± 6
57 ± 17
99 ± 41
58 ± 25
107 ± 25
145 ± 31
58 ± 17
93 ± 19
80 ± 15
116 ± 27
70 ± 28
102 ± 35
77 ± 18
51 ± 14

1349 ± 107
1430 ± 51
1423 ± 77
976 ± 54
1106 ± 59
924 ± 56
1216 ± 75
1339 ± 115
814 ± 71
1293 ± 95
1280 ± 63
2246 ± 70
2369 ± 68
1115 ± 92
1010 ± 91
1916 ± 97

Table 3.1: Fluorescence lifetime and anisotropy decay time constants as a function of anesthetic
concentration. The fluorescence lifetime data were fitted to a single exponential decay function
and the anisotropy decay data were fitted to a two-exponential decay component function.

42

Concentratio
n (mM)
Control
Pentobarbital

Isoflurane

Halothane

Dz (MHz)

0
1
2
5
7
10
2
3
5
7
10
2
3
5
7
10

1050 ± 330
3910 ± 1280
6190 ± 820
4300 ± 1010
2450 ± 740
4220 ± 1300
2270 ± 450
1660 ± 310
4210 ± 990
2620 ± 460
3060 ± 500
2120 ± 410
3540 ± 1030
2380 ± 640
3160 ± 620
4860 ± 1060

Dx (MHz)
124 ± 10
117 ± 5
117 ± 7
171 ± 10
151 ± 8
180 ± 12
137 ± 9
124 ± 12
205 ± 19
129 ± 10
130 ± 7
74 ± 2
70 ± 3
149 ± 14
165 ± 16
87 ± 5

Dz/Dx

DSE

ηDSE

8.5 ± 3
34 ± 12
53 ± 10
25 ± 7
16 ± 5
23 ± 8
17 ± 4
13 ± 3
21 ± 7
20 ± 4
24 ± 5
29 ± 7
50 ± 15
16 ± 5
19 ± 5
56 ± 15

(ps)
385
120
77
108
182
109
197
262
108
173
151
220
136
187
143
99

(cP)
7.1
2.2
1.4
2.0
3.3
2.0
3.6
4.8
2.0
3.2
2.8
4.1
2.5
3.4
2.6
1.8

Table 3.2: Dz, Dx, Dz/Dx, DSE and ηDSE values derived from experimental data as a function
of anesthetic concentration.

The data contained in Table 3.2 provide significant insight into the interactions of the three
anesthetics and the lipid bilayer. Before examining these details, however, it is instructive to
compare the calculated viscosity values for perylene reorientation in the bilayer structures as a
function of anesthetic identity and concentration (Figure 3.3). These data demonstrate three
important points. The first is that all of the anesthetics are interacting significantly with the acyl
chain region of the lipid bilayers which can be supported by the decrease in the viscosity of the
acyl chain region in these vesicles that were treated verse those that were not. The second point
of interest is that the viscosities are on the order of half what is measured for the same bilayer
that has not been exposed to anesthetics. This finding is consistent with a number of other
43

studies on lipid bilayer structures where the addition of constituents to the lipid bilayer typically
reduces the organization and thus the viscosity of the bilayer structure. The third point is that
there is a slight, qualitative trend in the data toward lower viscosities with increasing anesthetic
concentration vide infra. Again, this is not a surprising result and it is indicative of the amount
of anesthetic interacting with the bilayer being proportional to the amount of anesthetic in
solution. These viscosity changes are supported when the Cartesian components of perylene are
analyzed.

44

8
black = pentobarbitol
red = isoflurane
blue = halothane

control sample
(no anesthetic)

7

viscosity (cP)

6
5
4
3
2
1
0
0

2

4

6

8

10

anesthetic concentration (mM)
Figure 3.3. Dependence of lipid bilayer acyl chain region viscosity detected by rotational
diffusion measurements and calculated using Eq. 3.1, on the identity and concentration of
anesthetic present in the vesicle containing solution. Uncertainty in each data point is ± 0.5 cP.
It is clear from the data contained in Fig. 3.3 that the anesthetics are interacting with the acyl
chain region of the lipid bilayer structure. With that information in hand, the anesthetic identityand concentration-dependent values of Dx, Dz and Dz/Dx can be considered. These data are
presented in Figs. 3.4 – 3.6.

45

Dz

7

Dx, Dz (GHz)

6

Dx

5
4
3
2
1
00

2

4

6

8

10

pentobarbitol concentration (mM)
70
60

Dz/Dx

50
40
30
20
10
00

2

4

6

8

10

pentobarbitol concentration (mM)

Figure 3.4. Top: Dz and Dx (GHz) as a function of pentobarbital concentration. Bottom:
Dz/Dx as a function of pentobarbital concentration.

46

Dz

7

Dx, Dz (GHz)

6

Dx

5
4
3
2
1
00

2

4

6

8

10

isoflurane concentration (mM)
70
60

Dz/Dx

50
40
30
20
10
00

2

4

6

8

10

isoflurane concentration (mM)

Figure 3.5. Top: Dz and Dx (GHz) as a function of isoflurane concentration. Bottom: Dz/Dx as
a function of isoflurane concentration.

47

Dz

7

Dx, Dz (GHz)

6

Dx

5
4
3
2
1
00

2

4

6

8

10

halothane concentration (mM)
70
60

Dz/Dx

50
40
30
20
10
00

2

4

6

8

10

halothane concentration (mM)

Figure 3.6. Top: Dz and Dx (GHz) as a function of halothane concentration. Bottom: Dz/Dx as
a function of halothane concentration.

48

For all data sets, the addition of anesthetic increases the ratio Dz/Dx relative to that of the
control data. This finding, in conjunction with the data in Figure 3.3 pointing to a lower local
viscosity experienced by the chromophore when anesthetic is present implies that the increase in
Dz/Dx does not represent a restriction of the chromophore local environment about x-axis
rotation. Rather, these data imply the increased facilitation of rotational motion about the z-axis
as a function of added anesthetic. Thus the addition of anesthetic to the lipid bilayer is consistent
with a reduction in the order of the acyl chain region.
The data presented in Figures 3.4 – 3.6 also demonstrate an apparent discontinuity in the
region between ca. 3 and ca. 5 mM anesthetic.

The functional forms of these data are

reminiscent of data seen for perylene reorienting in DMPC lipid bilayers at a series of
temperatures, above and below the gel-to-fluid phase transition.

41

The implication of such

changes in Dz/Dx as a function of anesthetic concentration is that the anesthetic is interacting
with the lipid acyl chain region in such a way as to alter its organization. It is noteworthy that
the apparent change in lipid acyl chain organization occurs in the same concentration region for
all of the anesthetics examined. While there is not a currently accepted structure-function
relationship for the operation of anesthetics on bilayers, it appears that the function sensed by
perylene reorientation is remarkably similar for the three anesthetics examined, despite the
substantial structural differences between them.
The issue in question in this work is whether or not the action of anesthetics on lipid
bilayers is related to the ability of these compounds to induce characteristic anesthetic effects.
The primary issue is whether anesthetics interact with the bilayer directly or with transmembrane
species to produce the desired effect. In either case, the putative mode of action is the reversible
49

loss of neuronal cell function. The work presented here demonstrates interactions between the
lipid bilayer and the anesthetics that appear to be remarkably similar despite the structural
variation of the anesthetics, this is supported with the data that indicates the increase in acyl
chain viscosity. The viscosity change upon exposure to all anesthetics shows that in all cases the
anesthetics are interacting with the bilayers. The data that reveal the similarities between all
three are the figures showing Dz, Dx and Dz/Dx with breaks in behavior in the 0.3 – 0.4 mM
concentration range in all cases. These interactions are either directly responsible for the
mediating control over bilayer porosity or they alter bilayer-protein interactions which, in turn,
mediate transmembrane channel activity.

In either scenario, it is the anesthetic-bilayer

interactions that are central to the operation of the anesthetic.

50

CHAPTER 4
Conclusions
We have used fluorescence anisotropy decay measurements to interrogate the local
environment of lipid vesicle structures with composition intended to mimic the neuronal
membrane. We have found that the acyl chain region of these vesicles undergo a measurable
change in organization when exposed to varying concentrations of selected general anesthetic
agents. Our findings are consistent with Modern Lipid Hypothesis (1997) that anesthetics
interact with the lipid membrane. However we cannot make a claim about whether or not they
interact with transmembrane proteins imbedded within the membrane since proteins were not
incorporated in the membrane in this study. This finding does not exclude the possibility that the
anesthetics may also act on transmembrane proteins.

Interactions between anesthetics and the

plasma membrane gives rise to structural changes in the membrane that lead to increased
motional freedom for a chromophore imbedded within the membrane acyl chain region. This
change in structure implies increased disorder in the lipid acyl chain region, a factor that may
have a significant effect on the ability of transmembrane proteins to fold into their functional
forms. Any resulting changes in the function of transmembrane proteins in the membrane due to
induced changes in the membrane from anesthetic interactions may account for data suggesting
increased or decreased neurotransmitter release upon anesthetic administration in model
organisms. There remains, however, much to be learned about the mode of anesthetic action in
the CNS, but this body of data has provided some insight into the molecular-scale interactions
between anesthetics and lipid bilayer structures. Future work will explore further the
relationship, or absence of one, between anesthetic structure and lipid bilayer structural changes,

51

more biorelevant concentrations of anesthetics, and subsequently, the interactions between
anesthetics and transmembrane proteins.

52

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56