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DEFINING A NOVEL MECHANISM OF HYDROXYUREA ON
ERYTHROCYTES

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5/08 K:IPro}IAcc&PrelelRC/DateDue.indd

DEFINING A NOVEL ROLE OF HYDROXYUREA ON ERYTHROCYTES
By

Madushi Upendrika Raththagala

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHYLOSOPHY
CHEMISTRY

2008

ABSTRACT

DEFINING A NOVEL MECHANISM OF HYDROXYUREA ON ERYTHROCYTES

By

Madushi Upendrika Raththagala

Recent findings implicate the red blood cell (RBC) as a determinant in
physiological events that regulate the blood flow in the circulatory system. It is currently
known that circulating RBCs contain millimolar amounts of adenosine tri phosphate (ATP)
and release a fraction of that ATP under certain physiological stimuli. It is hypothesized
that oxidative stress and certain drug metabolites can affect the ability of the RBC to
release ATP and thereby have an effect on pathophysiological conditions in disease status.

A novel fluorescence based assay using monochlorobimane (MCB) in conjunction
with the standard addition method was developed to quantitatively determine glutathione
(GSH) levels in RBCs without any separation of GSH from the complex biological matrix.
The average GSH concentration of a 1% RBC solution was 0.042 i 0.002 mM for 6
rabbits, which translates to a cellular concentration of 362 d: 20 amol/RBC. The ability to
perform dynamic measurements provides a means to evaluate the effect of oxidant stressors
on RBCs. GSH concentrations were determined in RBCs subjected to the specific oxidant,
diamide, at 5 minute time intervals for up to 45 minutes. The results obtained from this
study greatly contributed to the establishment of the relationship between oxidant stress, a

weakened antioxidant system and RBC derived ATP release. The importance and necessity

of such a method is in developing microfluidic-based diagnostic tools, metabolomics
studies, and systems biology applications.

Subsequently, a novel micro flow based chemiluminescence technique was
employed to quantify and determine the effect of hydroxyurea, the currently approved
therapy for sickle cell disease (SCD), on ATP release from RBCs. The release of ATP from
RBCs increased from 0.350 :t 0.063 to 0.591 :I: 0.105 umol/l for n = 8 rabbits when these
RBCs were incubated for 20 min with 50 uM hydroxyurea. This observation was further
verified using varying concentrations of hydroxyurea (25-200 nM) and the highest ATP
release was observed when a 7% hematoctrit of RBCs incubated with 100 uM
hydroxyurea. Interestingly, the same trend of ATP release from RBCs was observed for a
7% hematoctrit of RBCs incubated in varying concentrations of spermine NONOate (125-
1000 nM) for 10 minutes. Further experimentation involved in the identification of the NO
source as well as defining the mechanism that exerts modulation of RBC derived ATP
release in the presence of hydroxyurea. It was determined that hydroxyurea was resulting in
an increased flux of calcium into the RBCs. These results suggested that the observed
modulation of ATP release from RBCs in the presence of hydroxyurea is essentially a NO
mediated effect. It is believed that NO produced within the RBC via NOS enzyme
modulates the RBC deformability, thereby facilitating the modulation of ATP release from
RBCS, accordingly. Finally, the development and utilization of novel analytical techniques
enabled the investigation of two currently interesting biochemical pathways in RBCs; the
effects of oxidant stressors and NO on RBC derived ATP release, which could ultimately
be useful in developing therapies that benefit the vascular complications associated with

diabetes mellitus and sickle cell disease.

Copyright by
MADUSHI U. RATHTHAGALA
2008

~ To my parents, Ruchira, and Netuli, with love

ACKNOWLEDGEMENTS

Finishing up this dissertation, memories of experiences I have had the over the
past four and half years flashes across my mind. I never imagined being on this path, but
today I’m grateful to those who inspired and directed me towards higher education.
Without them, I would not have achieved anything at all.

First and foremost my sincere gratitude is to my advisor Prof. Dana Spence for his
guidance and support through out my graduate studies. His enthusiasm, inspiration,
critical thinking and great efforts to explain things clearly, provided a good basis to the
content of this thesis. The first meeting with Dr. Spence changed the direction of my
whole scientific career, and I’m grateful for his genuine interest in research and
convincing me to choose analytical chemistry instead of Molecular Biology, which I
never thought I would do someday in my life.

I would like to thank the members of my committee at Michigan State University,
Prof. Gary Blanchard, Prof. John McCracken, and Prof. Gregory Baker. I greatly
appreciate the feedback I got from them and especially, I’m thankful to Prof. McCracken
for allowing me doing EPR experiments in his laboratory. Also, I’m grateful for many at
Wayne State University, who helped me in numerous ways during the first three years of
graduate school. I greatly appreciate the help I received from Prof. Christine Chow
during the recruiting process and the first few months in USA. Also, I would like to thank
Prof. Colin Poole and Prof. David Benson for being in my committee and for their

advices and comments on my project.

vi

Also, I my warm thanks to all the lectures at University of Colombo, Sri Lanka
for their support, encouragement and especially for helping me realize the importance of
pursuing graduate studies. I must thank my high school Zoology teacher, Mr. Damitha
Pinnagoda for directing me towards undergraduate studies in Chemistry.

Next, I would like to thank all of the past and present Spence group members for
helping me when ever I needed their strength. I warmly thank Dr. Paul Root for valuable
advice and friendly help. Thanks to Wasanthi, and Luiza for being good friends from the
very first day and sharing in every aspect of being a PhD student. Thanks to Ajith for
helping me in many ways over the last three months where I was stacked with lots of
experiments. Thanks to Wathsala for being a nice friend all the time. Thanks to Teresa,
Andrea, Nicole, Chia, Jen, Steve, and Adam for making the everyday tasks more
enjoyable. Most importantly, I will not forget the fun times we had in Spence group after
every oral, seminar and on B’days.

I cannot end without thanking my amazing friends. I’m really fortunate to have
friends like Imalka and Maheshi in my life. If it was not Maheshi’s effort, I would not
have come to US to pursue graduate studies. Imalka was there for me in every moment in
my undergraduate studies sharing lots of memories. Thanks to Manori, Sunithi, Janani,
and Rasika for being very nice friends every possible ways. Thanks to Wasanthi,
Krishanthi, and Chinthaka for making my life easier and enjoyable in US and I won’t
ever forget the long nights we had over the last four years.

Finally, the most important people in my life, my family. I would like to thank
my parents for their unconditional love and endless support. Special Thanks to my father

in believing in me and encouraging me to pursue my goals and dreams. I wish my mom

vii

was there to share the happiness with me today. I truly don’t know where I would be
without them in my life. I must thank to my loving sisters Miroshi, and Maheshi for their
love and support when ever I needed. Thanks to Ruchira for being by my side all the time
sharing my happiness and frustration equally and loving me so truly. Netuli, my cute little
baby girl, thanks for being a “happy baby” most of the time and understanding what a
student mom is really like. Also, I would like to thank my mother-in-law and cousin for
their help in nearly last 8 months. Without their help I would not have finished my thesis
this early.

Also, there are more other people who helped me in numerous ways and I wish to

express my warm and sincere thanks to all of them

viii

TABLE OF CONTENTS

LIST OF FIGURES ............................................................................ xi

CHAPTER 1 Red blood cell structure, function and clinical implications. . . . 1

1.1 Red blood cell characteristics ........................................................................ 1
1.2 membrane structure and deformability.............................................. 2
1.3 Metabolic pathways of RBCs ........................................................ 5
1.4 Antioxidant systems ofRBCs 8
1.5 Novel roles of RBCs in the vasculature ........................................... 10
1.5.1 ATP as a signaling molecule .............................................. 12
1.5.1.1 Mechanism of ATP release from RBCs ..................... 12

1.5.2 NO and RBCs .............................................................. 15

1.6 Red blood cells and its involvement in disease status ......................... 20
1.6.1 RBCs and diabetes mellitus .............................................. 21

1.6.2 RBCs and sickle cell disease ............................................. 24
References .................................................................................. 28

CHAPTER 2 Dynamic monitoring of glutathione in erythrocytes, without a

separation step, in the presence of an oxidant insult ..................... 39

2. 1 Glutathione ........................................................................... 39

2.2 Oxidative stress, glutathione and diabetes mellitus ............................ 43

2.3 Glutathione measurements: currently available methods and limitations....46
2.4 Fluorescence determination of GSH in RBCs: a novel method

without a separation step ................................................. 49

2.5 Experimental ........................................................................ 50

2.5.1 Generation of washed RBCs ............................................. 50

2.5.2 Methods and materials .................................................... 50

2.6 Results and discussion ............................................................. 52

2.7 Conclusions .......................................................................... 64

References .................................................................................. 66

CHAPTER 3 Modulation of erythrocyte derived ATP release in response to

3.1
3.2
3.3
3.4

3.5

hydroxyurea and nitric oxide ................................................ 71
Hydroxyurea in vasculature ....................................................... 71
Nitric oxide in vasculature ......................................................... 74

Effect of hydroxyurea and NO on erythrocyte derived ATP release. . . . . 77

Chemiluminescence measurement of deformation induced ATP release
from RBCs ........................................................................... 79
Experimental ........................................................................ 84

ix

3.5.1 Methods and materials .................................................... 84

3.5.2 Instrumentation ............................................................ 86
3.6 Results and discussion ............................................................. 87
3.7 Conclusions ........................................................................ 101
References ................................................................................. 1 02

CHAPTER 4 Effect of hydroxyurea on nitric production in erythrocytes: A novel

mechanism of hydroxyurea on sickle cell disease ....................... 108

4.1 Sickle cell disease and nitric oxide bioavailability ........................... 108
4.2 NO forming reactions of hydroxyurea .......................................... 111
4.2.1 In vitro NO formation from hydroxyurea ............................. 112

4.2.2 In vivo NO formation from hydroxyurea .............................. 114

4.3 A novel mechanism of hydroxyurea on erythrocytes ........................ 118

4.4 Experimental ....................................................................... 120
4.4.1 Methods and materials ................................................... 120

4.4.1.1 Chemiluminescence detection of ATP ..................... 121

4.4.1.2 AAS studies for calcium measurements in RBCs. . . . . 123

4.4.1.3 EPR for HbNO detection in RBCs .......................... 124

4.5 Results and discussion ............................................................ 126
4.6 Conclusions ........................................................................ 147
References ................................................................................. 148
CHAPTER 5 Overall conclusions and future directions ................................ 154
5.1 Overall conclusions ............................................................... 154

5.2 Future directions .................................................................. 159
References .................................................................................... 1 61

Figure 1.1

Figure 1.2

Figure 1.3

Figure 1.4

Figure 1.5

Figure 2.1

LIST OF FIGURES

Figure illustrating the size and the shape of human erythrocyte and the
membrane characteristics. Scanning electron micrographof human
erythrocyte shows the biconcave disc shape cell (a), while the
schematic representation of the RBC cytoskeleton shows the
attachment ofthe cytoskeletal proteins (b)3

Schematic representations of the important metabolic pathways in
RBCs. Glycolysis, pentose phosphate pathway, and the GSH
regeneration pathways are interconnected and dependent upon
NADPH and ATP production. Abbreviations are listed separately. . 6

Schematic representation of the effect of ATP released from RBCs on
the regulation of vascular caliber. The released ATP in response to
mechanical deformation of RBCs can stimulate NO synthesis in the
endothelial cell. The produced NO can diffuse into underlying smooth
muscle layer, thereby triggering the smooth muscle
relaxation ...................................................................... 11

The proposed mechanism of ATP release from human erythrocytes.
The first step of the mechanism is the activation of the G proteins in
response to certain physiological stimuli such as shear stress or
hypoxia. Then the cascades of events lead to the extrusion ATP
molecules from RBCs. Abbreviations: Gs and Gi are hetertrimeric G
proteins; PKA- protein kinase A; CFTR- cystic transmembrane
conductance regulator protein .............................................. 14

Figure illustrating the interaction and electron transport through the
cofactors, NADPH, FAD, FMN to haem and BH4 moiety via the
activation of calcium-calmodulin complex. Final product of the
synthesis ....................................................................... 18

Structures of reduced glutathione (GSH) and oxidized glutathione
(GSSG) ........................................................................ 40

xi

Figure 2.2

Figure 2.3

Figure 2.4

Figure 2.5

Figure 2.6

Figure 2.7

Figure 2.8

Schematic representation of the regulation of GSH levels in RBCs.
GSH synthesis is a two step ATP dependant enzymatic mechanism
that involves y-glutamyl cysteiny synthetase and glutathione
synthetase. GSH redox cycle involves three enzymes; GPx-
glutathione peroxidase, G6PD- glucose-6- phosphate dehydrogenase,
and GR- glutathione reductase .............................................. 42

Proposed mechanism of the involvement of increased oxidative stress
of RBCs nvascular complications in diabetes mellitus The deficiency
of G6PD activity decreases the intracellular NADPH levels thereby
affect GSH regeneration process. Lower intracellular GSH levels may
affect the RBC deformability, consequently the ATP release from
RBCs. The decreased amounts of ATP could affect the NO synthesis
from vascular endothelium leading to the vascular complications
associated with the disease45

GSH reacts with non fluorescent MCB to form the fluorescent MCB-
GSH conjugate ................................................................ 49

Initial attempts to quantify GSH in RBCs. The fluorescent intensity of
0.1% RBCs (a) were lower than MCB without RBCs (b).The
fluorescence intensity of MCB probe in 100 pM standard GSH (c)
was higher than the MCB probe in the presence of 0.1% RBC that was
prepared in a 100 pM standard GSH solution (d) .................. 53

Order of reagent addition for GSH determination using the method of
sanded additions.Varying volumes of water (730 — 750 pl) are added
to a standard cuvette, followed by 100 pl of a 1% RBC sample.
Next,100 pl of MCB and varying amounts of GSH (resulting in a 1.0
ml mixture) were added simultaneously, and the reagents were allowed
to react for 10 minutes prior to obtaining the fluorescence emission at
478 nm ......................................................................... 55

The standard addition method was used to quantify GSH in
erythrocytes by adding increasing volumes of 1 mM standard GSH

added to a mixture of 0.1% RBCs, 250 pM MCB and 2.5 U/pl GST to
make 4-20 pM final GSH concentrations, respectively. . . . . . 56

Calibration plot obtained for the standard addition method. The
fluorescent intensities were plotted against the volume of standard

xii

Figure 2.9

Figure 2.10

Figure 2.11

Figure 3.1

Figure 3.2

Figure 3.3

Figure 3.4

GSH solutions added to each RBCs sample. The resulting equation of
the line y = 0.651x + 3.08 (R2 = 0.9948) was used to determine the
GSH concentration in the RBCs ............................................ 57

The ability of MCB to monitor changes in reduced glutathione
concentration in erythrocytes was demonstrated upon the addition of
20 pM diamide to a 1% HCT sample of RBCs. The first bar in the
graph is the reading for no diamide added and thusepresents the
amount of GSH originally present before the addition of the xidant. As
expected, the GSH levels deceases immediately after the addition of
oxidant insult,but returned to the near initial afier about30 minutes...60

The effect of concentration of diamide on GSH regeneration ability
was demonstrated in 20 and 40 pM diamide concentrations. The black
bars represent the GSH levels of RBCs in 20 pM diamide, while gray
bars represents the GSH levels of RBCs in 40 pM diamide. GSH
regeneration ability was delayed in 30 minutes for 0.1% RBCs
incubated with 40 pM diamide compared to 0.1% RBCs incubated
with 20 pM diamide ........................................................... 61

A kinetic study demonstrating the importance of GST in quantifying
GSH in erythrocytes. The change of fluorescent intensity as a function
of time wavelength is shown for no GST and three varying GST
concentrations (0.5-2.5 U/ pl) ............................................. 63

Structure of hydroxyurea .................................................... 72

Schematic mechanism of the chemiluminescence generation from
luciferinand luciferase in the presence of ATP. Note the importance of
molecular oxygen in the generation of the intermediate ofexcited
oxyluciferin .................................................................... 82

A Schematic representation of the instrumentation used to quantify the
deformation induced ATP release from erythrocytes. RBCs are
pumped through two different silica microbore tubing where the RBCs
are deformed. The RBCs mix at the T junction emitting the light by the
chemiluminescence reaction ................................................ 83

Quantitative determination of ATP release from RBCs in the presence
of hydroxyurea using flow through chemiluminescence detection. The

xiii

Figure 3.5

Figure 3.6

Figure 3.7

Figure 3.8

black bar represents the ATP release from a 7% hematoctrit of RBCs
while the gray bar represents the ATP release from a 7% hematoctrit
of RBCs incubated with 50 pM hydroxyurea for 20 minutes. The two
bars are significantly different ((P< 0.03) and errorbars are reported as
SEM for n = 8 rabbits .......................................................................... 88

The effect of glybenclamide on ATP release from RBCs in the
presence of hydroxyurea. The gray bar represents the ATP release
from a 7% hematoctrit of RBCs incubated with 50 pM hydroxyurea
while the dark gray bar represents the ATP release from a 7%
matoctrit of RBCs incubated with 100 pM glybenclamide followed by
50 pM hydroxyurea. The value of (*) is significantly different from
others (P<0.01) and the error bars are reported as SEM for n = 5
rabbits .......................................................................... 90

The increased ability of hydroxyurea to release ATP from RBCs under
flow conditions. The black bars represent the ATP release from a 7%
hematoctrit of RBCs while the gray bars represent the ATP release
from a 7% hematoctrit of RBCs incubated with 50 pM hdroxyurea for
20 minutes. The bars corresponding to flow conditions (*) are
significantly different (P<0.001). However, the bars denoted as (**)
are different at P<0.05. Error bars were reported as SEM for n = 4
rabbits .......................................................................... 91

Chemiluminescence intensity change for RBC samples treated with
varying concentrations ofliydroxyures. Spectrum (a) is the
chemiluminesence intensity recorded for 7% hematoctrit of RBCs
reacted with luciferin-luciferase mix in the flow system while spectra
b, c, d, and e represent the 7% hematoctrit of RBCs incubated with
50,100,150, and 200 pM hydroxyurea, respectively. The signal
intensities were taken at a rate of 10 points/sec for 20 seconds time
period ........................................................................... 93

Effect of hydroxyurea concentration on deformation induced ATP
release from RBCs. Highest ATP release was observed for 7%
hematoctrit of RBCs incubated with 100 pM hydroxyurea for 20
minutes. After that, the ATP release started decreasing reaching to a
value that was not significantly different from untreated RBCs at 200
pM hydroxyurea. The values of (*) and (**) are significantly different
(P<0.02) and the error bars are reported as SEM for n =7 rabbits. 94

xiv

Figure 3.9

Figure 3.10

Figure 3.11

Figure 3.12

Figure 4.1

Quantitative measurement of deformation induced ATP release from
7% RBCs in the presence and absence of NO donor, spermine
NONoate. The black bar represents the ATP release for untreated
hematocrit of 7% RBC, while the gray bar represents the 100 nM
NONOate treated hematoctrit of 7% RBCs. n=5 and the error bars are
represented as SEM ......................................................... 95

Chemiluminescence intensity change for RBC samples treated with
varying concentrations of spermine NONOate. Spectrum (a) is the
chemiluminesence intensity recorded for 7% hematoctrit of RBCs
reacted with luciferin-luciferase mix in the flow system while spectra
b, c, d, and e represent the 7% hematoctrit of RBCs incubated with
100, 250, 500 nM spermine NONOate, respectively. The signal
intensities were taken at a rate of 10 points/sec for 20 seconds time
period ........................................................................... 97

Effect of spermine NONOate on deformation induced ATP release
from RBCs. The ATP release was initially increased gradually with
increasing concentration of NO donor and the highest release was
observed for 7% RBCs incubated with 250 nM Spermine NONOate.
Then, the ATP releasestarted decreasing gradually reaching to a point
at 1000 nM where the ATP release was not significantly different from
the initial untreated 7% RBCs. The two bars represented as (*) are
significantly different in P< 0.01 and the two bars represented as (***)
are significantly different in P<0.05. n = 4 rabbits and the error bars
are reported as SEM ......................................................... 98

The effect of cellular oxidant, diamide on RBC ATP release in the
presence of spermine NONOate. The black bar represents the ATP
release for hematocrit of 7% RBCs while gray bar with medium lines
represents the ATP release for 7% RBCs in 250 nM spermine
NONOate. Obviously, the lowest ATP release was found to the 7%
RBCs incubated with 20 pM diamide (dark gray bar). However, the
ATP release is not increased significantly for 7% RBC incubated first
with 20 pM diamide followed by 250 nM spermine NONoate. The (*)
and C”) are significantly different in P<0.001 for n =3 rabbits and the
error bars are reported as SEM .......................................... 100

Three general pathways that depicts the formation of NO from
hydroxyurea under in vivo or in vitro conditions (A). Proposed
mechanism of the formation of NO by hydroxyurea in the presence of
heme proteins (B) ........................................................... 113

XV

Figure 4.2

Figure 4.3

Figure 4.4

Figure 4.5

Figure 4.6

Figure 4.7

The reactions of HbNO formation by hydroxyurea from the reaction
of oxy, deoxy, and methemoglobinnThe reactions of hydroxyurea
with oxyhemoglobin and methemoglobin results in the formation of

nitroxide radical which further oxidizes to form molecular
NO ............................................................................. 115

An EPR spectrum showing the characteristic peaks for Fe2+-NO
complex at g = 6 and g = 2 ............................................................... 129

Typical EPR Spectra obtained from control RBCs (A) and 20 minutes
after 50 pM hydroxyurea treated RBCs (B). Spectrum C is the
difference obtained by subtracting B from A. The EPR settings were
as follows; power 5 mW, modulation further experimentation is
needed to draw a conclusion regarding the ATP release from RBCs in
response to the NO formed from hydroxyureafrequency 9.46 GHz;
field modulation amplitude 2.8 G, conversion time 327 ms, scan
average 4........... ............................................................................. 130

Typical EPR Spectra obtained from control RBCs (a) and 100 pM
spermine NONOate treated RBCs (b). Spectrum (c) is the difference
obtained by subtracting (b) from (a). The EPR settings were as
follows; power 5 mW, modulation frequency 9.46 GHz; field
modulation amplitude 2.8 G, conversion time 327 ms, scan average
4............ ......................................................................................... 131

The difference spectra obtained for spermine NONOate treated RBCs
and hydroxyurea treated RBCs. The triplet hyperfine structure at g
=2.01 is the characteristic signal of HbNO in RBCs. The triplet
hyperfine structure corresponding to the g values of HbNO in NO
treated RBCs is not significant in hydroxyurea treated RBCs. The EPR
settings were as follows; power 5 mW, modulation frequency 9.46
GHz; field modulation amplitude 2.8 G, conversion time 327 ms, scan
average 4 ..................................................................... 132

Typical EPR spectra obtained for the calibration curve of NO treated
RBCs. A: untreated RBCs. Spectra B,C, and D were obtained from
RBCs treated with 20, 40 and 100 pM spermine NONOate
respectively. The EPR conditions were as follows: power, 5mW;

xvi

Figure 4.8

Figure 4.9

Figure 4.10

Figure 4.11

Figure 4.12

frequency 9.46 Ghz; sweep time, 15 minutes; scan average,
4 ............................................................................... 133

Calibration curve of HbNO signal obtained from the reaction of RBCs
and gaseous NO. The peak heights of the HbNO signals in figure 4.8
are plotted against the varying NO concentrations of gaseous NO (20-
100 pM). R2 = 0.998 ......................................................... 132

Measurement of ATP release from RBCs in the presence of L-NAME
and hydroxyurea. RBCs incubated with L-NAME resulted in the
lowest release of ATP from RBCs (third bar) showing the importance
of intra erythrocyte NO production on ATP release. Hydroxyurea did
not have any effect on ATP release in the presence of L-NAME
indicating an important function of hydroxyurea on NOS in RBCs.
The values between (*) and (**) are significantly different (P< 0.04). n
= 4 rabbits and the error bars are represented as SEM ................. 135

Measurement of the ATP release from RBCs in the presence and
absence of calcium ions in the medium. Black bars represent the ATP
release of untreated RBCs and gray bars represents the ATP release
from RBCs treated with 50 pM hydroxyurea. Increased ATP release is
observed for hydroxyurea treated RBCs in PSS while no significant
increase of ATP release from RBCs is observed for RBCs treated with
hydroxyurea in the calcium free PSS. The data represented as (*) and
(**) are significantly different in P< 0.01. n = 5 rabbits and the error
bars are reported as SEM .................................................. 137

Atomic absorption spectroscopic study of calcium quantification in
RBCs. The first bar (gray) represents the calcium concentration in the
cytoplasm of hematocrit of 10% RBCs. The second bar (black)
represents the calcium concentration in the cytoplasm of hematocrit of
10% RBCs incubated with 50 pM hydroxyurea solution for 20
minutes. The values are significantly different in P< 0.025. = 7
rabbits and the error bars are represented as SEM ...................... 139

Typical EPR Spectra obtained from control RBCs (a) and 20 minutes
after 50 pM hydroxyurea treated RBCs in normal PSS (b). Spectrum
(c) is the difference spectra obtained by subtracting (b) from (a). The
EPR settings were as follows; power 5 mW, modulation frequency
9.46 GHz; field modulation amplitude 2.8 G, conversion time 327 ms,
scan average 16 .............................................................. 140

xvii

Figure 4.13

Figure 4.14

Typical EPR Spectra obtained from control RBCs (a) and 20 minutes
after 50 pM hydroxyurea treated RBCs in calcium free PSS (b).
Spectrum 0 is the difference spectra obtained by subtracting (b) from
(a). The EPR settings were as follows; power 5 mW, modulation
frequency 9.46 GHz; field modulation amplitude 2.8 G, conversion
time 327 ms, scan average 16 ............................................. 141

The difference EPR spectra obtained from RBCs incubated with 50
pM hydroxyurea in calcium free PSS (a) and in calcium containing
PSS (b). The triplet hyperfine structure with g values at 2.02 indicates
the presence HbNO in spectrum (b). However, the corresponding
peaks of HbNO are not significant in spectrum (b). The EPR settings
were as follows; power 5 mW, modulation frequency 9.46 GHz; field
modulation amplitude 2.8 G, conversion time 327 ms,scan average
16 .............................................................................. 142

xviii

CHAPTER 1

Red blood cell, structure, function and clinical implications

1.1 Red blood cell characteristics

Erythrocytes, commonly referred to as red blood cells (RBCS), are the most
common type of blood cell in a vertebrate’s vascular system.1 RBCs are highly
specialized cells specifically designed for the purpose of oxygen transport from the
environment to the respiratory tissues in the body.

Human RBCs are small, biconcave disc shaped cells. The average mean volume
of RBC is ~ 82-95 fl while the diameter ranges from 6-8 pm.2‘ 4 Human RBCs occupy
45% of the total blood volume and carry 4.5-6.5 x 106 of RBCs in 1 pl of blood.5 These
cells are much smaller than the average cell in the body, although the biconcave shape
results in the highest possible area for a given volume allowing for a shorter diffusion
path for oxygen. Moreover, mature human RBCs lack nuclei and organelles such as
mitochondria and golgi bodies and therefore are incapable of synthesizing their own
proteins, lipids and nucleic acids.6 All of these modifications are essentially deigned to
provide enough space for the oxygen carrying protein, hemoglobin and an average RBC
contains about 270,000 million hemoglobin molecules or 28-34 g of hemoglobin per d1 of
RBCs. In this respect, RBCs have often been considered (and oversimplified) as a “bag of

hemoglobin” designated only for oxygen transport throughout the body.

In reality, RBCs contribute to many important steps in regulatory mechanisms of
cellular functions other than their major role of oxygen transport in the body. They do
carry important enzymes and substrates for several metabolic pathways and antioxidant
defense mechanisms.7 These processes are essential for RBC survival, as well as for the
maintenance of the mechanical properties of the RBC. Indeed, the human RBC travels
through the circulatory system about 500,000 times during its life time of ~120 days.8
Therefore, the RBC membrane is also specially designed to withstand the mechanical
stress placed on it within the circulation. Maintenance of RBC deformability is necessary
for its proper functioning and is dependant on essentially three factors; internal viscosity,

the geometry of the cell and the RBC membrane deformability.9

1.2 RBC membrane structure and deformability

The RBC membrane consists of a lipid bilayer and an underlying skeletal protein
network (cytoskeleton). The skeletal network of the RBC membrane is a dense,
filamentous hexagonal shaped protein network, directly laminating the inner leaflet of the
lipid bilayer via the interaction of integral proteins present in the bilayer.'0’ H The major
component of the cytoskeleton is spectrin, which is a rod like protein composed of two
non identical subunits of 240 kD (a sub unit) and 220 kD ([3 sub unit) intertwining side
by side.'2‘ ‘3 The a and B subunits attach side to side in an anti parallel manner forming
the heterodimer. The tetrameric structure formed by these two heterodimeric proteins is
the predominant form in the cytoskeleton and attaches to several other proteins to form

the skeletal protein network. Spectrin predominantly interacts with actin proteins

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membrane characteristics.lo Scanning electron micrograph of human erythrocyte shows
the biconcave disc shape cell (a).'4 Schematic representation of the RBC cytoskeleton
shows the attachment of the cytoskeletal proteins (b).15

creating the spectrin-actin lattice like network underlying the lipid bilayer. Actin is
present in oligomers of 12-20 actin filaments, and the ratio of actin to spectrin in the
cytoskeleton consists of one actin oligomer to six spectrin tetramers. The attachment of
the spectrin-actin lattice to the membrane bilayer is through the protein ankyrin.'6’ ’7 RBC
ankyrin is an asymmetric globular protein of 215 kD that contains binding sites for both
spectrin and the anion transporter protein (band 3) in the bilayer. Band 3 is a glycoprotein
of ~93,000 bp and is the most abundant protein of the human RBC membrane.15 It is
important in the transport of chloride and bicarbonate across the membrane. In addition to
interacting with ankyrin, band 3 has a high affinity for RBC membrane glycolytic
enzymes, as well as to hemoglobin molecules in the cytoplasm.18

Protein 4.1 in the cytoskeleton helps the tight binding of spectrin and actin, which
are otherwise very weak. It is composed of four major domains and has shown intriguing
multifunctional structural properties by attaching vertically and horizontally to the lipid
bilayer and the skeletal network. Protein 4.1 is also a component of the ternary complex
that comprises the junctional complex of the cytoskeleton.'9’ 20 In addition, there are
several other accessory proteins associated with the cytoskeletal structure and,
collectively, all of these molecules provide RBC shape, stability and deformability.

The ability of RBCs to change geometry under the influence of mechanical forces
and revert back to their original shape is important for proper functioning of the cell.
Therefore, the structure and deformability of the RBC membrane has been the subject of
many investigations for many decades due to their importance in blood rheology. The

proteins of the cytoskeleton has arranged in a definite way that preserves the geometry of

the cell while allowing a temporarly shape change when traveling through the arterioles

and capillaries. Failure to maintain some level of deformability is often associated with
rheological complications or in other words microcirculatory disturbances leading to
several pathological conditions, such as hemolytic anemia, sickle cell disease,
sperocytosis and diabetes mellitus.9‘ 21’10’ 22 Therefore, a systematic study of deformation
and flow, provides insight into some remarkable properties of the RBC that are associated

with its own unique structure and pathological consequences.
1.3 Metabolic pathways of RBC

Oxygen transport is a vital process in all living systems. In humans, hemoglobin
has specialized functionality and structure to transport oxygen to skeletal muscles.6
However, RBCs also contain hundreds of other organic and inorganic molecules in the
cytoplasm in order to perform different fimctions for their survival. Some of these
functions comprise generation of metabolic energy (ATP) and reducing agents (NADH
and NADPH).23 RBCs need energy to maintain osmotic balance and volume (via the
many ATP dependant ion pumps), replenish the adenine nucleotide pool, and maintain
membrane deformability and to protect cells through anti oxidant defense mechanismsz’
7’ 8 All of these functions require the existence of several metabolic pathways including
glycolysis, the pentose phosphate pathway (PPP), the 2,3, diphosphoglycerate shunt and
nucleotide metabolic pathways.23

Among them, glycolysis is the predominant metabolic pathway present in the
RBC for producing ATP. Glucose is the main energy source or the starting point of the

pathway and it produces 2 ATP and 2 NADH molecules per glucose via anaerobic

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glycolysis. The lack of mitochondria in the RBC prevents glucose from being oxidized
aerobically through the citric acid cycle; hence the final products of glycolysis are lactate,
ATP and NADH. The PPP contributes to the redox status of the cell by producing 2
NADPH molecules per glucose molecule.24 Glucose is converted to glucose-6-phosphate
by glucose-6-phosphate dehydrogenase (G6PD), a key regulatory enzyme in the RBC
PPP. The major function of the PPP is to produce ribose sugar for nucleotides and to
maintain the continuous supply of NADPH.2 NADPH is used as an obligatory substrate
for glutathione maintenance and the PPP is the only way of producing NADPH in RBCs.
One molecule of NADPH is produced by reducing a NADP+ and is used as a hydrogen
donor molecule for the reducing reaction of glutathione reductase. The activity of the
GSH redox cycle is dependant upon the activity of G6PD, and healthy cells maintain the
reducing environment within the cells by properly maintaining the glutathione
antioxidant status and the PPP. It is believed that under normal circumstances only 1/60
of the G6PD activity is utilized, but when the oxidant stress is high than the normal
G6PD activity is increased according to the oxidant stress placed on the cells.

Any abnormality of the RBC metabolic pathways may lead to serious disease
conditions, as these metabolic functions are interconnected via different steps and
conditions in the RBC. However, our current understanding of the relationship between
the metabolic deficiencies of RBCs and disease pathogenesis is lacking. Recent research
is focused on generating a quantitative overview of metabolism and its regulation through
a systems biology approach where all the factors involved for certain problems are
evaluated.25 Therefore, rapid detection methods, simultaneous analysis of different

metabolites, and miniaturization are key steps to consider.

1.4 Antioxidant systems of RBCs

Oxidative stress, a pronounced pro-oxidant state, is the increased production of
reactive oxygen species or the decrease in the reducing capacity of cellular redox couples
(or weakening of the antioxidant systems).26’ 27 The destructive effects of the stress are
determined by the extent of the imbalance resulting from the production of reactive
species and detoxification ability of the cellular system. Reactive oxygen species, which
include free radicals and peroxides, are formed in various cellular reactions. Increased
lipid peroxidation, protein oxidation and DNA damage through reactive oxygen species
or impairment of antioxidant defense systems in cells have lead to many pathological
conditions including diabetes mellitus, atherosclerosis, Parkinson’s disease, and
Alzheimer’s disease.”30

Antioxidants are the cells’ foremost supply of protection against various reactive
oxygen species that include free radicals and peroxides. Presence of a working
antioxidant mechanism is crucial for the protection of enzymes, membrane proteins and
lipids from excessive oxidants and free radicals, as well as to maintain hemoglobin in its
ferrous form without being oxidized. Erythrocytes are prone to higher oxidative damage
due to the exposure of high oxygen content, thereby necessitating maintenance of their
structural and functional proteins via an antioxidant defense mechanism.31 Disturbance of
the redox balance by the production of reactive species such as hydroxyl, superoxide,
peroxinitrites, alkoxy or peroxyl radicals and peroxides will lead to RBC membrane

damage, death or many pathophysiological consequences. Cellular components in RBCs

such as lipids, proteins and enzymes can be exposed to reactive species, thus making

them susceptible to oxidative damage.31 Among them, thiol containing proteins (eg.,
spectrin) are subjected to oxidation via its cystein thiols.l4 Disulfide bonds can be formed
in the presence of oxidants and this could lead to the destruction of important membrane
properties. Also, maintaining hemoglobin in the reduced state is essential for gas
exchange and transport. Some hemoglobin molecules also attach to the cell membrane,
hence, is important to maintain RBC mechanical properties. Therefore, it is a prerequisite
to have an antioxidant system in cells, especially in RBCs.

The most abundant non-enzymatic antioxidant found in the RBC is the reduced
form of glutathione (GSH).32 This tri-peptide thiol (y-L-glutamyl-L-cysteinyl glycine)
acts as a major bio reducing agent via its high electron donating capacity linked to the
sulfliydryl (-SH) group. GSH is present in millimolar concentrations in most mammalian
cells, which makes it the most concentrated intracellular antioxidant in the RBC.” 34
GSH exists in two forms, reduced (GSH) and oxidized (GSSG). In healthy cells, the
GSSGzGSH ratio is 1/ 10, however, modification of this physiological ratio is often an
indicator of a disease state that may result in various pathological events. The proper
GSSGzGSH ratio is maintained through tight regulation of GSH synthesis and the GSH
redox cycle. Glutathione reductase converts oxidized glutathione back to its reduced form
at the expense of NADPH produced via the PPP. Another GSH redox cycle enzyme,
glutathione peroxidase, is also important in removing hydrogen peroxide from cells. It
reduces H202 to H2O while oxidizing GSH to GSSG. G6PD is an important redox cycle
enzyme, as it is important for the activity of glutathione reductase by supplying the

NADPH pool. The combined effects of GSH redox cycle enzymes maintain the reducing

environment within the cell, and thereby protect RBC characteristics within its activity of

120 days.

1.5 Novel roles of RBCs in the vascular system

1.5.1 ATP as a signaling molecule

It has become increasingly clear that, in addition to functioning as an oxygen
transporter in the body, RBCs have other roles that can be considered important steps in
the regulation of many cellular functions. One such role is its ability to release adenosine
triphosphate (ATP), which has been identified as a potential regulator of vascular
resistance or vessel diameter.6‘ 21‘35’ 36 ATP, which is released from the erythrocyte into
the circulation, can bind to P2Y purinergic receptors present in the vascular endothelial
cells resulting in the synthesis and release of nitric oxide (NO).37'39 NO synthesized in
endothelial cells can be released both abluminally and luminally. When released
abluminally, NO can interact with the vascular smooth muscle cells, stimulating vascular
relaxation, and thereby an increase in vascular caliber.38

The relationship between NO synthesis in the vascular endothelium in response to
RBC-derived ATP has not been established until 1995 when Sprague et al. reported on
the role of the RBC as a determinant in vascular resistance.40 It is generally accepted that
increased shear stress is the major stimulus for endothelial NO production.4| However,
Sprague et al. showed that alterations in the shear stress did not stimulate NO synthesis in
isolated perfused rabbit lungs in the absence of rabbit RBCs.40 In other words, they

concluded that the RBC is a determinant of endothelial derived NO synthesis.

10

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In 1996, Sprague et a1. further demonstrated the possible mechanism that links RBCs to
NO synthesis.36 They proposed that the ATP is released from RBCs in response to
mechanical deformation and evokes the synthesis and release of NO from the endothelial
cells lining the pulmonary circulation.” 42’ 43 This hypothesis was supported by several
previous findings. First, ATP is a signaling molecule and stimulates NO synthesis.“
Second, ATP is the major adenine nucleotide present in the cell and is present in
millimolar concentrations in RBCs. Third, it has a recognized role in signaling pathways
of other cells.

Several analytical methods have been employed to determine the concentration of
deformation induced ATP release from RBCs. In filtration methods, the RBCs are forced
through small pores of filters, collected and assayed for ATP release. Although, this
method of producing deformation induced ATP was successful in early applications, it
has some limitations that prevent on-line measurement of ATP release from RBCs.44 The
new yet simple technique developed by Spence et al. enabled the detection of ATP
release from RBCs upon passage through microbore tubing.45 Moreover, this method
resulted an improved understanding of the mechanisms that regulate vascular resistance
under in vivo conditions as this method mimics closely the in vivo physiological

conditions found in the circulation.

1.5.1.1 Mechanism of ATP release from RBCs

Red blood cells release ATP in response to certain physiological stimuli such as

reduced oxygen tension or hypoxia, membrane deformation and a decrease in pH.46‘47 It

12

has also been recently reported that RBCs release increased amounts of ATP in the
presence of pharmacological stimuli.48 Low nanomolar levels of ATP are sufficient for
the activation of purinergic receptors present in the endothelial cells, and thereby may
regulate the vascular tone.43 However, ATP is a large, charged molecule and is unable to
cross the membrane through diffusion. Therefore, the presence of a specific mechanism
that induces ATP release to the extracellular environment is necessary for the efflux of
ATP from the cell.49

To date, the key components required for the release of ATP from RBCs have
been identified. In initial studies, it was determined that ATP crosses the membrane via
the ATP binding cassette (ABC), an ion channel protein. Next, the cystic fibrosis trans-
membrane conductance regulator protein (CFTR), which is indeed an ABC family
protein, was identified as the protein required for ATP release from erythrocytes.50 The
finding that CFTR protein activity was a requisite for the signaling mechanism of ATP
release from cells led to further characterization of the pathway.
Specifically, it is now well established that heterotrimeric G protein (Gi,)49‘ 5‘
adenylyl cyclase,52 protein kinase A (PKA) and CFTR are important components of the
signal transduction pathway of RBC-derived ATP release.46‘49‘S3 The first step of the
pathway is the receptor mediated activation of G protein in response to either
physiological stimuli such as mechanical deformation, pH or hypoxia or pharmacological
stimuli such as iloprost or mastoparan.49 The first step of the ATP release mechanism is
the activation of G proteins. Catalytic activity of adenylyl cyclase can be stimulated by
both Gas and Gui protein subtypes. For example, the heterotrimeric protein Gm], Gun and

Gun subunits have been identified in human and rabbit RBC membranes

13

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14

related to ATP release from RBCs.49 Activation of the specific sub component of the G
protein stimulates adenylyl cyclase present in the membrane and thereby increases
intracellular concentrations of 3’-5’ cyclic adenosine mono phosphate (CAMP) within the
cell. Adenylyl cyclase is important in PKA activity and it is indeed important in the
phosphorylation of CFTR, and thereby initiating the translocation of ATP
extracellularly.54 However, to date, research to reveal the protein that is responsible for
the ATP extrusion is still undergoing. It is either the direct involvement of the CFTR

protein or another protein that is activated upon CFTR phosphorylation.

1.5.2 NO and RBCs

Nitric oxide is a paramagnetic, heterodiatomic free radical that exists as a gas at
room temperature. It is one of the 10 smallest molecules in nature, making it one of the
most rapidly diffusible molecules. The diffusion coefficient (D) of NO is approximately
3300—3800 umz/s in aqueous solutions and also it can rapidly diffuse through membrane
structures due its solubility in hydrophobic phases.55

The Nobel Prize for medicine or physiology was awarded to Robert Furchgott,
Louis Ignarro, and Ferid Murad in 1998 for identifying NO as the endothelial-derived
relaxing factor. Since then, this small free radical has been identified in a variety of
beneficial and detrimental physiological roles through direct and indirect reactions due to
its complex chemistry. 56

Several important physiological roles of NO in biological systems including

cardiovascular, immune, and nervous systems have been identified. It is well established

15

that NO is involved in vascular homeostasis by regulating the vascular resistance, smooth
muscle relaxation and inhibiting RBC and platelet adhesion in the circulatory system.57'58‘
59 NO synthesized in the endothelium affects 20-30% of basal human blood flow.60

However, there has been considerable debate over the interaction and
physiological role between endothelium-derived NO and RBCs as well as the
involvement of RBC—derived NO in the vascular function.61 For example, a considerable
amount of NO synthesized from the endothelium interacts with blood components known
to have an effect on vascular homeostasis. However, it has long been known that NO
reacts very fast with hemoglobin in RBCs; in fact hemoglobin is a scavenger of NO in
biological systems. Initially, the high reaction rates of hemoglobin with NO have taken
away the potential significance of NO on RBCs. However, the observation of varied
reaction times for free hemoglobin as compared to hemoglobin encapsulated in RBCs
suggested a possible role of NO in RBCs.62 The erythrocyte membrane and the unstirred
plasma layer around the RBC create a diffusional barrier to NO, thereby preserving the
reactivity within circulation.63 However, for free hemoglobin, the reaction rate is only
dependant on the diffusion rate of NO to the heme pocket. NO reacts with
oxyhemoglobin to form methemoglobin and nitrate, and with deoxyhemoglobin to form
iron—nitrosyl-hemoglobin.6O It is accepted that the rapid reaction of the heme group of
hemoglobin and NO produce a heme-iron nitrosyl adduct (Hb(FeNO)), although no
bioactivity is found upon this formation.64

Several researchers have shown that NO bioactivity is maintained through the

formation of nitrosothiols.65‘66 Biochemical studies confirm that S-nitrosylation at 2

cysteine residues of the [3 subunit of hemoglobin is conserved in mammals and birds. S-

16

nitrosohemoglobin (SNOHb) is formed under oxygenated conditions of Hb(FeNO) where
heme —to cys transfer occurs.64 This finding was supported by the fact that NO may be
transported as SNOHb in erythrocytes. SNOHb is known to be associated predominantly
with the RBC membrane in humans, and more specifically, is bound to the cysteine
residues in the hemoglobin-binding cyt0plasmic domain of the anion exchanger AEl .67

It has been reported in the literature that RBCs can serve as oxygen responsive
transducers of vasoconstrictor and vasodilator activity and this is partly due to NO
bioactivity.64 It has also been shown in the literature that SNOHb is formed upon
oxygenation of (Hb(FeNO)) by heme to cysteine NO transfer, and transfer from low mass
— nitrosothiols.64’68‘69 Continuous cycling of hemoglobin between oxygenated and
deoxygenated states enables the rapid binding and release of NO. It is known that formed
SNO-Hb is a stable in the oxygenated state of hemoglobin, but gains vasodilator potency
upon deoxygenation of hemoglobin. However, the detection of SNOHb and other NO
metabolites in biological systems is challenging due to its presence in low concentrations
and the instability of the species. The inconsistent findings of NO derived species have
also fueled the arguments over the physiological importance of these species.57 Despite
the debate over the “real” transport form, SNOHb formation is proposed way of
maintaining and transporting the bioactivity of NO in the vascular system.

NO is produced by the nitric oxide synthase enzyme (NOS) in cells. NOS utilizes

the amino acid L-arginine, converting it into citrulline and No.70 The enzyme requires

17

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several cofactors such as FAD, FMN, tetrahydrobiopterin, and heme.72 All of these
cofactors are needed for the electron transport through the two domains. NOS exists in
three different isomers commonly known as endothelial NOS (eNOS or NOS III),
neuronal NOS (nNOS or NOS 1) and inducible NOS (iNOS or NOS 11) based on location
and activity.”75 For example, eNOS is present predominantly in the endothelial cells,
while nNOS is found in neuronal tissues. In contrast, iNOS can be seen in wide variety of
tissues and is synthesized upon stimulation. iNOS and eNOS can be categorized as
constitutive NOS as these isoforrns are important in maintaining the basal NO levels in
tissues. Since the activation of these two isofonns requires the formation of the calcium-
calmodulin complex, the flux of calcium in the cytosol is the starting step of the synthesis
of NO.

Human RBCs consist of both inducible and constitutive forms of NOS genes and
thus are capable of producing their own NO and modulating several functions.76 Also, it
has been suggested that RBC derived NO has a contributory role in maintaining RBC

77 RBC derived NOS activity depends on the intracellular Ca2+

physiological behavior.
levels and phosphorylation at serine 1177 regulated by the phoshatidylinositol—3-kinase
(P13K) where it resembles eNOS in some ways. This enzyme is functionally active even
at basal levels of calcium and thereby maintains RBC deformability and supports RBC
passage through capillaries.78 Considering the facts of NO synthesis, it is possible that
both extracellular and intracellular sources of NO can affect cells in the circulatory
system. Several researchers have suggested that NO is effect on RBC deformability is

modulated by both endothelial and RBC derived NO, but the exact mechanism of the

effect of NO on RBC deformability is still not clear.79 It has been shown that NO

19

inhibitors have an effect on erythrocyte deformability in septic shock.” 81 Also, NO
donors such as spermine NONOate increase the cell deformability. In addition, it has
been found that erythrocyte deformability was influenced by polymorphonuclear
leukocytes through a mechanism involving NO release.82 It is also known that NO can
modulate (either increase or decrease) erythrocyte deformability within a certain range of
NO concentrations, as stated previously. In contrast, it was reported that NO inhibits
hypoxia-induced ATP release from erythrocytes. Sprague et al. reported that NO
released luminally from endothelial cells may function in a negative feedback manner to
inhibit ATP release from erythrocytes.38 They further believed that this inhibition occurs
through a G protein activated pathway.”84 However, these findings have not taken the
attention away from the potential positive effects of NO and its effect on RBCs in the
microcirculation.

The clinical implications of RBC NOS activity is evolving and therapeutic
interventions to enhance NO production via enhancing NOS activity has been considered
in therapies for sickle cell disease, heart failure, and ischemia-repurfusion injury.
Conversely, suppressing this activity is a better therapeutic approach for sepsis, where
higher NO production stiffens the membrane and inhibits the RBC passage through the

capillaries.”

1.6 Red blood cells and its involvement in disease status

Even though RBCs have been subjected to many investigations since its

recognition as an oxygen carrier, little attention has been given to their involvement

20

indisease status. However, recent studies have demonstrated the effect of metabolic
deficiencies or membrane abnormalities of RBCs on clinical complications of several
diseases, where the major cause of the disease is not thought to be RBC related. Diabetes
mellitus, pulmonary hypertension, cystic fibrosis and sickle cell disease are some of the
diseases that fall into this category. Therefore, the study of RBC and its relevance in

disease pathogenesis is vital for the advancement of medicine and drug discovery.

1.6.1 RBCs and diabetes mellitus

Diabetes mellitus, commonly known as diabetes, is a chronic disease of
disordered metabolism, resulting in abnormally high glucose levels in the blood stream.
Diabetes affects 23.6 million people or ~ 8% of the United States population.

The cause of hyperglycemia or high glucose levels in the blood is due to a
combination of hereditary and environmental factors. The known basis of the disease is
either the loss or diminished production of the insulin hormone by the pancreatic B cells
or the decreased response or sensitivity of insulin to insulin receptors on the cell
membrane. The net result of poor insulin usage is reduced intake of glucose by the cells,
thereby increasing the concentration of glucose in the blood stream. Long term
complications of diabetes include retinopathy, nephropathy, neuropathy, and vascular
diseases. Hypertension, increased risk of stroke and heart failure are often associated with
people suffering from diabetes.

Since 1921, diabetes has been treated and controlled by externally supplying the

insulin hormone. To date, research has been centered on insulin alone for the treatment of

21

diabetes. Nevertheless, diabetes is a chronic disease with no cure that requires short term
and long term management to prevent other complications associated with the disease.
However, in recent years, research has involved attempts at better understanding the role
of RBCs in diabetes and its complications.

Oxidative stress is a major component of molecular, cell and tissue damage in a
number of diseases including diabetes. Numerous recent studies have shown that patients
with diabetes mellitus have increased oxidative stress and an impaired antioxidant
defense system.” 85 Free radicals can react with proteins, lipids, proteins, carbohydrates,
etc, thereby affecting the overall physiology of the living system.86

Oxidative stress in diabetic RBCs is known to be higher in comparison to the
healthy RBC. Moreover, diabetic-induced oxidative damage is higher in RBCs in
comparison to other tissues in the body, largely because RBCs are carriers of oxygen.
Also, the RBC is known to have a higher content of iron and polyunsaturated fatty acids,
which are prone to oxidative damage.87 Diabetic RBCs are subjected to a high glucose
environment within the circulation and this hyperglycemic condition leads to non-
enzymatic protein glycation in RBCs, especially of hemoglobin and other membrane
proteins.88 It has been shown that the interaction of glucose with proteins can form
glycosylated proteins through the spontaneous chemical reaction of glucose with amino
groups of the proteins.” 90 Moreover, glucose is prone to auto oxidation that can produce
highly reactive oxygen species as well as other charged molecules. All of these molecules
can damage the RBC itself. Schwartz et al. have shown that spectrin, ankyrin and protein

4.2 are the most glycosylated RBC proteins.88 Interestingly, all of these proteins are

22

components of the RBC cytoskeleton, which provides evidence for the genesis of the
decreased deformability of diabetic RBCs. It is well established that type 2 diabetic RBCs
are less deformable in comparison to RBCs obtained from healthy humans.” Many
studies have indicated that one aspect of diabetes is the abnormal rheological properties
of RBCs.88 These abnormalities may be due to the decreased deformability of the
membrane, RBC cytoplasm viscosity and increased erythrocyte aggregation.
Collectively, all of these properties suggest that protein glycosylation and increased
oxidative damage results in decreased deformability of RBCs. However, there are other
features of the RBC that may result in oxidative damage in diabetic RBCs that need, to be
considered. One of these features is a weakened antioxidant system.

A weakened antioxidant system has been observed in patients with diabetes. It has
been reported that GSH metabolism is altered in both type 1 and type 2 diabetes patients.
This is supported by findings that chronic hyperglycemia increases the activity of the
polyol pathway and free radical generation, which leads to increased GSH oxidation.
Moreover, activation of the aldose reductase pathway reduces NADPH concentrations
thereby diminishing GSH regeneration.86 It is well established that GéPD, a key
regulatory enzyme in the PPP, is deficient in the RBCs of diabetic patients.28 Because
NADPH is produced exclusively via the PPP in RBCs, a deficiency of G6PD diminishes
the availability of NADPH for glutathione regeneration, ultimately resulting in a
decreased activity of the GSH antioxidant defense system (which can further affect the
overall oxidative stress in diabetic RBCs).32

Erythrocytes, via their ability to release ATP in response to physiological stimuli,

have been shown to be a determinant in maintaining vascular resistance. Carrol et al. has

23

shown that diabetic RBCs release lower amounts of ATP when compared to the RBCs
obtained from healthy humans.858prague et al. reported that the RBCs obtained from type
2 diabetic patients have reduced expression of the Gi protein. Gi is an important
component of ATP release from RBCs and reduced expression of this protein leads to
deceased production of CAMP, which subsequently leads to less RBC-derived ATP
release.54 This suggests that defects in RBC physiology may affect the vascular reactivity,
the major complication associated with diabetic patients.92

Taken collectively, increased oxidative stress, an impaired antioxidant system,
and hereditary factors such as deficient enzyme/protein activities may lead to
pathophysiological complications associated with diabetes and it is important to have a
better understanding of these mechanisms for a better therapeutic approach to disease
management. However, until recently, no attempts were taken to demonstrate the
relationship among the antioxidant system, enzyme deficiencies and the ATP release
from RBCs. Carrol et al. and Subasinghe et al. provided evidence for the first time
showing the importance of antioxidant systems on ATP release from RBCs.93
Importantly, this work enabled a quantitative determination of ATP release due to the

disturbance of the antioxidant system.

1.6.2 RBCs and sickle cell disease

Sickle cell disease (SCD) is an autosomal recessive disease affecting more than

75,000 people in the US. It is the most common genetic disease affecting African-

Americans (0.15% are homozygous and 8% have the sickle trait).94 The characteristic

24

feature of this hereditary disease is a single amino acid mutation of the [3 subunit of the
hemoglobin molecule. Normal hemoglobin (HbA) consists of 2 a globin and 2 B globin
chains. The substitution of glutamate to valine results in a new type of hemoglobin called
hemoglobin S (HbS). Formation and the presence of the HbS in the RBC results in the
majority of pathophysiological conditions associated with the disease.“ 95

Patients with SCD suffer from a wide range of major clinical manifestations,
including acute episodes of pain, stroke, acute chest syndrome, leg ulcers and organ
damage.96 These RBCs when traveling through the microvasculature, are subjected to
large shape changes under deoxygenated conditions giving rise to the “sickle” shaped
cells. The primary reason of sickling is the polymerization of the HbS molecules in the
RBC under deoxygenated conditions. Formation of sickled cells is the critical point in
vaso occlusive crisis, which is the main complication associated with the disease. Under
deoxygenated conditions, these HbS molecules can polymerize into long fibers thereby
increasing the RBC rigidity. High rigidity or decreased deformability leads to vascular
obstruction and ischemia. Moreover, these cells have a shorter life span (16 days)
compared to healthy RBCs (120 days) and are prone to hemolytic destruction (lysis) due

to decreased deformability.” 98

Also, the damaged cell membrane results in increased
adhesion to the endothelium enhancing the chances of vaso occlusion crisis, stroke and
pulmonary hypertension.99 Finally, activation of endothelial cells, platelets, and the
coagulation system leads to clinical implications associated with the disease. These
factors make the patients anemic and often in need of blood transfusions. As a result,

SCD is considered a chronic rheological disease and a significant contributor to blood

flow failure.

25

Even though the molecular basis of SCD has been known for decades, there was
no promising therapy until 1995. The revolutionary publication of the critically important
therapy was a breakthrough in the management of SCD. Hydroxyurea, a ribonucleotide
reductase inhibitor, is a proven treatment for sickle cell disease.100 The widely accepted
mechanism of action of hydroxyurea is its ability to induce fetal hemoglobin (HbF)
production in RBCs. However, the exact mechanism of increased HbF production by
hydroxyurea is still not clear. Moreover, the beneficial effects of hydroxyurea do not
always correlate with the increased HbF production, suggesting there may be an
additional mechanism of hydroxyurea in SCD patients.

The improvement of rheological properties including increased whole cell
deformability, reduced red cell density and improved erythrocyte hydration status have
been observed with patients receiving hydroxyuream' However, the mechanism of action
of hydroxyurea in relation to improved rheological properties still remains to be
elucidated, especially regarding the improvements of vascular properties, although
various mechanisms have been proposed in the recent literature.

Even though SCD was originally considered a genetic disease, its major
pathophysiologycal complications are associated with the alteration of vascular
properties. SCD patients are known. to have stiffened RBCs, low NO bioavailability,
increased oxidative stress, increased RBC- endothelial interactions, all of which can be
categorized under the impairment of one or more metabolic functions of the RBCs.
Therefore, an understanding of the RBC properties, especially the factors that affect the

regulation of blood flow, would be beneficial for patients having SCD. Moreover,

26

defining the mechanisms hydroxyurea in SCD would be helpful in developing novel

therapies to overcome the clinical complications associated with the disease

27

10.

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38

CHAPTER 2

Dynamic monitoring of glutathione in erythrocytes, without a separation

step, in the presence of an oxidant insult

2.1 Glutathione

Glutathione (L-y-glutamyl-L-cysteinyl-glycine) is the most abundant, non
enzymatic, low molecular mass, thiol-containing tripeptide found in virtually all the
mammalian cells, plants and microorganisms."2 Its facile electron donating capacity,
linked to the sulfliydryl (SH) group of cysteine has made glutathione a potent antioxidant
and a convenient cofactor in living systems.3 Glutathione is present in millimolar
concentrations (1-10 mM) inside the cell cytosol (SS-90%) or other aqueous phases,
although the actual concentrations are varying depending on the cell type.

Glutathione exists in two forms. The antioxidant, or reduced form, is
conventionally known as glutathione (GSH) while the oxidized form is known as
glutathione disulphide or GSSG. The GSH molecule is made up of three amino acids,
glutamate, cystein and glycine, covalently linked end to end by the peptidic y linkage.
GSSG is formed by two GSH tri-peptides linked by a disulfide bond. The intracellular
glutathione is in the reduced state, which represents ~90% of the total glutathione content
in the cells. The oxidized concentrations are generally low but can be increased under

various oxidative stresses or pathological conditions.

39

 

 

 

 

 

 

O O O O O O
\C/ \C/ \C/
CH2 CH2 CH2
NH NH NH
020 0:0 020
H2 H2 H2
HC---C -—SH HC—C —S——S—C —CH
NH NH NH
CH2 CH2 CH2
0 /C\ /C\
y \ - / o- -o \
O O O
O
(GSH) (GSSG)

Figure 2.1 Structures of reduced glutathione (GSH) and oxidized glutathione (GSSG)

GSH/GSSG is the major redox couple that determines the antioxidant capacity of the cell.
The ratio of GSHzGSSG has been shown to be an indicator of oxidative stress and
therefore can be used as an effective measure of a diseased state.5

The most important function of glutathione in red blood cells (RBCs) is to
detoxify the reactive oxygen species, free radicals and reactive elecrophiles.5 Being the

most abundant antioxidant in RBCs, GSH converts into an oxidized form (GSSG) while

40

reducing the various oxidants present in the cells. However, oxidized GSH or GSSG must
revert back to its reduced form to maintain its activity. The conversion of GSSG to GSH
is known as GSH regeneration.l Glutathione reductase is necessary for the regeneration
process in the presence of NADPH, a cofactor produced in the pentose phosphate
pathway. Importantly, this pathway is the only means of producing NADPH in the
erythrocyte for glutathione regeneration.

GSH is synthesized from L-glutamate, L-cysteine, and glycine in two ATP-
dependant pathways. The first step is the synthesis of y-glutamylcysteine from L—
glutamate and L-cysteine via the activity of y-glutamylcystein synthase enzyme. Next, a
glycine residue is added to the C-terminal of y-glutamylcystein by the enzyme
glutathione synthase. Even though GSH synthesis is the only source of glutathione for
cells, the intracellular GSH concentrations are dependant on both de novo synthesis of
GSH and the activity of the GSH redox cycle.

The GSH redox cycle is composed of several enzymes, namely, glutathione
peroxidase (GPx), glutathione reductase (GR), and glucose-6-phosphate dehydrogenase
(G6PD).6 GPx is involved in neutralizing H202 to H2O while oxidizing the GSH to
GSSG. However, GSSG is then converted back to GSH by the enzyme GR. G6PD is
important in the generation of the NADPH cofactor for the GSH regeneration process.
The GSH redox cycle is a tightly regulated process and helps protect the cell from
excessive oxidative damage. In addition to the GSH/GSSG redox couple, there are
several other redox couples that maintain the reducing environment of the cell. However,
the GSH redox status is important in maintaining the steady state value of the

intracellular redox potential as GSH exists in 100-1000 folds higher than the reduced

41

L-glutamate + L-cystein

v-glutamyl cysteinyl

v-glutamyl cysteinyl synthetase

Glycine
GSH synthetase

GSH
*boz NADPH

GPx GR G6PD

*ho NADP‘
GSSG

Figure 2.2 Schematic representation of the regulation of GSH levels in RBCs. GSH
synthesis is a two step ATP dependant enzymatic mechanism that involves y-glutamyl
cysteinyl synthetase and glutathione synthetase. GSH redox cycle involves three
enzymes; GPx- glutathione peroxidase, G6PD- glucose-6-phosphate dehydrogenase, and
GR- glutathione reductase

42

form of other redox couples.Modifications of the GSH cycle or enzyme levels may result
in an imbalance of the antioxidantzoxidant ratio leading to cellular structure fragility or
various pathophysiological conditions. In addition to its function as an antioxidant or
antitoxin, GSH is involved in diverse biological processes in living systems including
protein synthesis, enzyme catalysis, transmembrane transport, cell metabolism.5
Numerous studies have associated alterations in RBC GSH concentrations with various
diseases including diabetes mellitus,7'9 Alzheimer’s disease,'0 HIV,ll multiple sclerosis'2

”"4 In all of these disease conditions, deleterious imbalances

and several types of cancers.
between the production and removal of reactive oxygen species and free radicals, as well

as depleted GSH levels have been observed. Therefore, the redox balance between GSH

and GSSG, or GSH regeneration, is important in maintaining cellular homeostasis.

2.2 Oxidative stress, glutathione, and diabetes mellitus

RBCs are subjected to higher oxidative stress compared to other cell types due to
their continuously high levels of intracellular oxygen and high iron contents. GSH is
known to be the most concentrated antioxidant found in RBCs and acts as the redox
buffer maintaining iron in its reduced state for hemoglobin function and fighting against
oxidative stress to maintain cellular deformability. In other words, RBC GSH levels are
crucial in maintaining the RBC’s ability to overcome oxidative stress-induced conditions
during the RBC’s lifetime of 120 days.ls

As mentioned above, higher oxidative stress has been linked to many diseases

including diabetes mellitus.”[7 A weakened oxidant defense mechanism, higher

43

production of oxidant species, and the decreased deformability of diabetic RBCs have
been known to exist for decades, but no study linked the oxidant stress of RBCs to the
complications associated with diabetes mellitus. A weakened antioxidant system is often
the result of decreased activity of glucose-6-phosphate dehydrogenase (G6PD), while the
higher production oxidants are an effect of an increased rate in protein glycation by
glucose. In addition, the decreased RBC deformability of diabetes may be due to the
oxidation of membrane proteins such as spectrin in the presence of oxidants.

It has been previously reported that RBCs are a required component for
maintaining vascular resistance via their ability to release adenine triphosphate (ATP), a
known stimulus of NO. The amount of ATP released from RBCs is dependant on the
deformability of RBCs, therefore the vascular complications associated with diabetes
include a higher occurrence of strokes, heart attacks and the high probability of having
hypertension. However, linking a physical characteristic of RBCs (e.g; RBC
deformability) to a weakened oxidant system is difficult as it requires experimental
methods to quantify the physiological consequence of oxidant insult placed on the RBCs.
Quantifying the GSH levels upon oxidative stress would be the first step in such a
determination as GSH levels represent the overall oxidant status of cells. However,
currently available methods for GSH quantification require separation of the cellular
constituents prior to measurement. These procedures are time consuming and therefore,
measuring an immediate physiological response from the RBCs upon oxidant insult
cannot be determined. Therefore, the objective of this study is to develop a new technique
to quantitatively determine GSH levels in RBCs under oxidant stress in order to

understand the effects of GSH depletion on disease diagnostics and disease pathogenesis.

44

  
   

G6PD

G6P———’ 6GP
fl

NADP NAD H

GSSG—Gi’ GSH /'

 
 
 
  
 

Figure 2.3 Proposed mechanism of the involvement of increased oxidative stress of
RBCs on vascular complications of diabetes mellitus. The deficiency of G6PD activity
decreases the intracellular NADPH levels thereby affect GSH regeneration process.
Lower intracellular GSH levels may affect the RBC deformability, consequently the ATP
release from RBCs. The decreased amounts of ATP could affect the NO synthesis from
vascular endothelium leading to the vascular complications associated with the disease. 18

45

2.3 Glutathione measurements: currently available methods and limitations

Numerous methods for glutathione measurements in different cell types have been
described in the literature. Early attempts to measure GSH in various cell types included

2

enzymatic,'9’ 0 colorimetric and fluorometric based assays. These methods employed
potent glutathione reductase inhibitors, such as N-ethylmaleimide for enzymatic assays,
and several probes such as 5-5’-dithiobis-(2-nitrobenzoic) acid (DTNB or Ellman’s
reagent), monochlorobimane (MCB) or monobromobimane (MBB) for fluorometric
assays. However, many of these methods have insufficient detection limits and low
precision that limit biological sample analysis.

Recently, several new techniques couple the separation and detection techniques
to quantify intracellular GSH in different cells and tissues. High performance liquid
chromatography (HPLC)2’2"23 and capillary electrophoresis (CE)24 are widely used to
quantify GSH or GSHzGSSG in biological samples. HPLC coupled to

”‘29 and ultra violet (UV) absorbancem'33 detection

electrochemical,”26 fluorescence,
methods are currently in wide use for the determination of GSH. Usually, UV
measurements are taken after derivatization with DNTB or with 2,4-dinitropyenyl
derivatives.31 Fluorescent measurements are carried out after separating GSH using
HPLC and then derivatized with several reagents such as monobromobimane (MBB)?”
ortho-phthalaldehyde (OPA), l-dimethylaminonapthalene-5-sulphony1 chloride (dansyl
chloride),28‘34 or ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate (SBD-F).2

Moreover, HPLC can be coupled to mass spectrometric analysis.”36 Mass spectrometric

detection is known to be much more sensitive compared to HPLC with UV, and HPLC

46

with fluorescence detection. More recently, CE with electrochemical detection, laser
induced fluorescence detection, and UV detection were reported to be a highly sensitive
method of quantifying GSH or GSHzGSSG in cellular systems.15 ’ 3 7' 3 8

While these methods have been successful in measuring GSH and, in the case of
those employing HPLC and CE, the GSHzGSSG ratio, each method has limitations.
Generally, one of the more challenging areas of biological analysis is overcoming matrix
effects. Large numbers of molecules in smaller volume, resembling structural features of
different molecules and instability of the molecules have complicated biological sample
analysis.

For example, methods that employ DNTB are often limited by its lack of
specificity for GSH. Fluorescence based assays using bimane derivatives in erythrocytes
have required the separation of cellular constituents. Those methods employing HPLC
and CE have provided the separation necessary to quantify GSH in biological samples.
GSH is a water soluble compound, thus extraction of GSH from biological matrices is a
challenge without employing a derivatization step. Also, the chemical instability of the
molecule, especially the susceptibility of the thiol group to oxidation, renders the
quantitative determination even more difficult.

Several analytical techniques have been developed to quantify GSH levels in
RBCs. However, wide differences in GSH concentrations in RBC samples have been
reported in the literature and are probably due to the various sample preparation and
analysis methods.” Improper sample preparation, collection, pre-treatment and storage of
the sample for long periods of time can cause large variations of the GSH content in

cells.”40 Moreover, the ability of GSH to undergo autooxidation prevents quantifying the

47

actual amounts of GSH inside the cells. Acid treatment is often carried out for RBC lysis,
but this may be harmful for actual quantification as the GSH oxidation diminishes the
GSH content in cells leading to possible underestimated values in the literature.15 Sample
preparation by acid treatment of proteins can lead to precipitation of oxyhemoglobin,
oxygen release and free radical generation, thereby altering the GSH redox cycle.
Recently, Zinellu et al. described a new method in which GSH oxidation was minimized
by using cold water for RBC lysis followed by membrane filtration for the separation of
cellular constituents.15 However, this method is not successful in large sample analysis
especially for diagnostic purposes because of the high expense in using membrane filters.
In summary, all techniques described in the literature employ either a separation method
or a derivatization step that results in a complicated analysis.

Quantitative determination of GSH is extremely difficult in RBCs compared to
other cell types because of their complex matrix, particularly in the presence of
hemoglobin in RBCs. A single RBC contains about 200 — 300 million hemoglobin
molecules or 11-18 g/dl of hemoglobin content. Hemoglobin absorbs light ranging from
the UV to the visible, preventing spectroscopic determination of different analytes in the
RBC. Therefore, the separation of hemoglobin from the RBCs is necessary for most
analytical techniques that employ UV and fluorescence detection. Taken collectively,
improvements in proper sample preparation techniques and detection techniques are vital
in quantifying GSH in biological samples.15 Moreover, the determination of GSH in
erythrocytes can potentially benefit clinical diagnosis at the early stages of disease.
Therefore, a rapid method with appropriate detection limits and high reproducibility is a

necessity for the analysis of biological sample.

48

2.4 Fluorescent determination of GSH using standard additions

Considering the drawbacks and limitations of the currently available methods of
quantifying intracellular GSH, a new fluorescence based assay was developed to quantify
GSH in erythrocytes. The primary objective of developing a new method is to have a
rapid, reliable and more sensitive way of quantifying GSH in erythrocytes without
separating GSH from other cellular constituents.4 The necessity of having such a method
is its utilization in disease diagnosis, metabolomics studies and microfluidic/lab on a chip
based applications.

The new technique employs the fluorescent probe, MCB in combination wih the
standard addition method. The use of MCB as a fluorescence probe for GSH

determinations has been previously reported.“42

 

 

 

 

 

 

 

 

 

CH3 CH3
0 O

\ GSH /S\ H \ S\‘

N CHZCI : N GSH
O O
/ CH3 / CH3
CH3 CH3
MCB MCB-GSH conjugate

Figure 2.4 GSH reacts with non fluorescent MCB to form the fluorescent MCB-GSH
conjugate

49

However, when used in conjunction with the method of standard additions to overcome
the complex matrix of the erythrocyte, it is possible to perform a quantitative
determination of the GSH levels in cells.4 The method of standard addition is a powerful
technique when the sample matrix is difficult or impossible to duplicate. This method is
beneficial for RBC studies since it compensates for complex matrix interferences. The
standard addition method employed in this assay was validated by the linear relationship
obtained between the response and the analyte of interest.

Importantly, this new method can be performed without any sample preparation
beyond isolation of the RBCs from the whole blood. Moreover, because no physical
separation of GSH from the cell matrix is required, this method can be used to determine
the GSH redox status in a dynamic manner when performed in a continuous flow stream.4
Thus, this technique enables a constant monitoring of a small portion of the overall

cellular metabolism in real time.

2.5 Experimental

2.5.1 Generation of Washed Red Blood Cells

RBCs were prepared on the day of use. For obtaining rabbit RBCs, male New
Zealand White rabbits (2.0-2.5 kg) were anesthetized with ketamine (8.0 mg/kg) and
xylazine (1.0 mg/kg) followed by pentobarbital sodium (15 mg/kg iv). After tracheotomy,
the rabbits were mechanically ventilated (tidal volume 20 ml/kg, rate 20 breaths/min;
Harvard ventilator). A catheter was placed into a carotid artery, heparin (500 units, iv)

was administered, and after 10 min, animals were exsanguinated. Blood was collected

50

into vials and the RBCs separated from other formed elements and plasma by
centrifugation at 500 x g at 4 °C for 10 min. The supernatant and buffy coat was removed
by aspiration. Packed RBCs were resuspended and washed three times in PS8 [PSS; in
mM; 4.7 KCl, 2.0 CaCl2, 1.2 MgSO4, 140.5 NaCl, 21.0
tris(hydroxymethyl)aminomethane, and 11.1 dextrose with 5% bovine serum albumin,

pH adjusted to 7.4].4

2.5.2 Methods and materials

Fluorescence Determination of GSH using Standard Additions

The standard addition method was employed to perform quantitative
determinations of GSH. The GSH standards were prepared by combining 100 pl of a
1.0% hematocrit of RBCs and varying amounts (0, 4.0, 8.0, 12.0, 16.0, and 20.0 ul) of a
1.0 mM stock solution of standard GSH (Sigma) in separate vials. Also added to the
mixture to aid in the labeling of the MCB probe was 50 ul of 100 units ml'lofglutathione
-S- transferase, (Sigma). The GSH stock was prepared by dissolving 0.0307 g of GSH
(Sigma) in 18.0 M!) distilled/deionized water (DDW). Next, varying volumes of buffer
were added to each vial such that, upon addition of 100 pl of 250 uM MCB (Molecular
Probes), the final volume would be equal to 1 ml. After the addition of the MCB to the
RBC/GSH/GST//buffer mixture, a 10 minute incubation period was allotted to allow the
MCB to react with GSH. Following the incubation period, the GSH-MCB fluorescence
was measured (ex. 370 nm, cm. 478 nm) for each of the prepared vials. The fluorescence

intensity was plotted as a function of volume of GSH stock added to each vial.4

51

Monitoring GSH upon Oxidant Attack

GSH was monitored in both static (non-flow) and dynamic (continuous flow)
systems. To demonstrate the ability of MCB to monitor the GSH redox status in
erythrocytes following an oxidant attack in the absence of flow, a 20 uM diamide
solution (Sigma) was added to 5000 uL of RBCs (1% hematocrit) to intentionally oxidize
GSH to GSSG.43 A stock solution of 2 mM diamide was prepared by dissolving 0.0344 g
of diamide in 100 ml of DDW. 100 pl of RBCs were removed every 5 minutes and used
to prepare samples containing a 0.1 % hematocrit of RBCs, GST, and MCB. The changes
in fluorescence were monitored every 5 minutes for up to 90 minutes (except for the first
reading; this reading was taken at 10 minutes because the MCB-GSH reaction was given

a minimum of about 10 minutes before a signal was measured).

2.6 Results and Discussion

Following the 10 minute incubation period, the MCB fluorescence in the presence of
RBCs was measured (ex. 390 nm, em. 520 nm) and found to be lower than the
fluorescence of MCB without any RBCs (figure 2.5 a, b).4 The RBCs contain GSH thus,
it was expected that the fluorescence signal would be higher in the presence of RBCs and
lower when MCB was measured in the absence of any GSH source. Samples (1000 ul)
containing 250 uM MCB and 100 uM GSH were prepared with and without 100 pl of
1.0% hematocrit of RBCs (figure 2.5—c, d). Again, the samples that contained RBCs

resulted in fluorescence emission that was lower than samples without any RBCs.4

52

 

140

 

ILLILJ

a
............... b
: -..-..-.. d
100{
3
8O - ’ITx
: ’-’ \\

Fluorescence intensity

 

 

 

 

450 500 550 600 650

Wave length (nm)

Figure 2.5 Emission spectra of the initial attempts of quantifying GSH and MCB. The
fluorescent intensity of 0.1% RBCs (a) were lower than MCB without RBCs (b). The
fluorescence intensity of MCB probe in 100 uM standard GSH (c) was higher than the
MCB probe in the presence of 0.1% RBC that was prepared in a 100 uM standard GSH
solution(d).4

53

It was suspected that the high molar extinction coefficient of hemoglobin at 390
nm was interfering with the excitation of the MCB. In fact, the molar absorptivity of
oxyhemoglobin at 390 nm and 370 nm are 167,748 cm'lfM and 88,176 cm'1/M,
respectively. This data, and the literature values, imply the interference of RBC
hemoglobin in the fluorescent measurement of GSH with MCB. Therefore, by changing
the excitation wavelength of MCB from 390 nm to 370 nm, the absorbance of
hemoglobin could be significantly decreased while still providing adequate excitation of
the probe.

Standards were prepared as described above and incubated for 10 minutes prior to
acquiring the fluorescence spectra (ex. 370 nm, em. 478 nm). GSH was quantified from
the calibration curve (figure 2.8) using the peak emission intensities shown in figure 2.7.
The GSH concentration in the RBCs were then determined from the y-intercept (b)
multiplied by the concentration of the GSH stock solution of 1 mM (Csmck) divided by the
product of the slope (m) and volume of RBCs added to each standard that was measured
(vied) (1).“

GSH RBC: bC stock/mVRBC (1)

Preparing the standards directly in the complex matrix of the sample, the hallmark
feature of the standard addition method, allowed for the quantitative determination of
GSH in erythrocytes without separation of cellular constituents or any sample
pretreatment.4 The GSH concentration per RBC from the data in figure 2.7 was
determined to be 0.047 i 0.003 mM.4 The overall standard deviation was calculated using

the standard deviations about the slope and intercept, respectively. This value is within

54

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55

 

 

20 q
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400
Figure 2.7

   
   
    

 

20 uM GSH

 

12 “M GSH

8 11M GSH

1% R805

 

 

450 500 550 600

Wavelength (nm)

The standard addition method was used to quantify GSH in erythrocytes

by adding increasing volumes of 1 mM standard GSH to mixture of 0.1% RBCs, 250 uM
MCB and 2.5 U/ul GST to make 4-20 uM final GSH concentrations, respectively.4

56

 

18‘
165
14-j

a 1

“a :

:12.

m r

d-l .

.E -

010:

Q -1

C :

8 8:

U) s

a) s

8 q

.J
2 6‘
u. :
4'.
1
2:
1
0 I l W [7'FT11lYIl

 

 

 

o 5 1o 15 20 25
Volume of GSH (pl)

Figure 2.8 Calibration plot obtained for the standard addition method. The

fluorescent intensities were plotted against the volume of standard GSH solutions added
to each RBCs sample. The resulting equation of the line y = 0.651x + 3.08 (R2 = 0.9948)
was used to determine the GSH concentration in the RBCs.4

57

the range of concentrations published for GSH in mammalian erythrocytes but are
somewhat higher than the previously reported for human RBCs.44 By incorporating the
volume of the RBC (87 fl), it was also determined that the amount of GSH per cell was
~400 amol. The average GSH concentration found in the bulk sample for the RBCs from
n=6 different rabbits was 0.042 :1: 0.002 mM. This number translates to a cellular
concentration of 3.99 :t 0.25 mM/RBC in the sample and 362 i 20 amol/cell.4 The
precision in these results are represented as a standard error of mean.

In order to provide evidence that the signal measured was actually due to the
MCB reacting with GSH, and not other thiols found in the cellular matrix, a sample was
spiked with a known amount of authentic GSH prior to preparing the standards used in
the standard addition determination.4 The amount of GSH found in the RBC sample was
0.046 mM. However, upon addition of 0.50 umoles of authentic GSH to the sample it
was determined that 99.8% percentage of the spiked GSH was recovered. These results
suggest that standard addition method can be employed to successfully determine GSH
levels in the RBCs without any sample preparation steps other than separating the RBCs
from the whole blood. Also, these results suggest that the MCB probe is specific for GSH
in these studies and that the fluorescence signal is due to the formation of the MCB/GSH
fluorescence emission.

GSH functions as the most abundant non-enzymatic antioxidant in RBCs. As
such, it is thought to protect other proteins from forming extensive oxidation products.
Once subjected to an oxidant insult, the RBC attempts to maintain normal levels of GSH
through GR-catalyzed reduction of the GSSG back to the protective GSH form. This

dynamic reduction-oxidation process is continuous in cells. Therefore, it is imperative

58

that a system by designed that enabled quantitative monitoring of the redox status of the
molecule of interest in the cell (GSH).

To demonstrate the ability of MCB to monitor the glutathione redox status in
erythrocytes following an oxidant attack, 100 ul of 2 mM diamide were added to 5 ml of
RBCs (1 “/0 hematocrit) to intentionally oxidize GSH to GSSG. Diamide is useful in
studying the GSH redox cycle because it is known to oxidize GSH specifically at a higher
rate than the other thiols.45 Aliquots of RBCs (100 pl) were removed every 5 minutes
and used to prepare 1 ml samples containing 0.1% hematocrit of RBCs and 250 uM
MCB. The changes in fluorescence were monitored every 5 minutes for up to 90
minutes. Figure 2.9 shows the initial decrease in fluorescence due to the GSH being
oxidized to GSSG in the RBCs. However, after approximately 20 minutes, the GSH is
slowly started regenerating due to the RBCs ability to maintain proper intracellular
oxidant status.18 The decrease in fluorescence emission exemplifies the ability of the
diamide to oxidize the GSH to the dimer. Importantly, the emission profile also shows the
ability of the RBC to return to levels of GSH that are statistically equivalent to the
original values prior to oxidant insult.

However, when the oxidant insult is higher (40 uM), the regeneration ability was
delayed by about 30 minutes compared to 20 uM diamide (figure 2.10). This observation
was further validated by using a higher concentration of diamide (100 nM). In that
instance, the ability of GSH to regeneration was lost due to the high oxidant stress. These
studies clearly demonstrate that the GSH levels are dynamic and redox cycle changes in
response to oxidants. Therefore, these results can be used in hypothesizing the exact

mechanism of action of GSH under oxidative stress

59

 

Fluorescence intensity
.3;
l

 

 

0 10 15 20 25 30 35 40 45 60 90

Time (min)

Figure 2.9 The ability of MCB to monitor changes in reduced glutathione
concentration in erythrocytes was demonstrated upon the addition of 20 uM diamide to a
1% HCT sample of RBCs. The first bar in the graph is the reading for no diamide added
and thus represents the amount of GSH originally present before the addition of the
oxidant. As expected, the GSH levels deceases immediately afier the addition of oxidant
insult, but returned to the near initial after about 30 minutes.

60

 

Fluorescence intensity
4:
H

1LLA44LA¥JJJL

 

 

0 10 15 20 25 30 35 40 45 60 90

Time ( min)

Figure 2.10 The effect of concentration of diamide on GSH regeneration ability was
demonstrated in 20 and 40 uM diamide concentrations. The black bars represent the GSH
levels of RBCs in 20 uM diamide, while gray bars represents the GSH levels of RBCs in
40 uM diamide. GSH regeneration ability was delayed in 30 minutes for 0.1% RBCs
incubated with 40 uM diamide compared to 0.1% RBCs incubated with 20 uM diamide.

61

Moreover, the results shown in figure 2.9 and 2.10 greatly contributed to our
understanding of the effect of oxidant stress on the RBC ATP release ability. The change
of GSH levels and ATP release from RBC were observed in the presence of diamide and
followed the same trend for over 45 minutes. Specifically, there was a rapid decrease of
both GSH levels and the ATP release from RBCs after 15 minutes of incubation with
diamide. These results also suggest that the reduced ATP release observed for diabetic
RBCs may be due to the weakened antioxidant systems found in diabetic patients. When
the RBCs are subjected to oxidant stress, the deformability of the RBC decreases
resulting in reduced ATP release from RBCs under mechanical deformation.
Collectively, this study was the first demonstration of the relationship between the
antioxidant oxidant status in RBCs and the ability of RBCs to release ATP, a known
stimulus for NO production from endothelium.18

This assay employed the glutathione-S-transferase (GST) enzyme. In order to
demonstrate the use of GST in the assay, a kinetic study was carried out with MCB and
GSH (figure 2.11). The function of GST is to catalyze conjugation reactions of GSH with
different electrophiles present in the cells. This enzyme is important in detoxification
reactions due to its ability to conjugate sulfydryl groups with other free radicals and
reactive oxygen species. In this assay, the use of GST increases the reaction rate of MCB
and GSH in RBCs allowing lower incubation time with the fluorophore. The average
time of incubation of MCB with GSH is ~18 min, but in the presence of 2.5 U/ul of GST
enzyme, the incubation time was decreased by 50%. This is important in evaluating the

redox status of GSH upon oxidant attack as GSH regeneration can be observed within 20

minutes of incubation time with diamide. Furthermore, this assay can be employed in

62

 

50

 

------ No GST
— — - " 0.50 U/ul
40 L 1.25 U/ul
— ........ 25 U/lll ’ ’

Fluorescence intensity

 

 

 

 

Time (seconds)

Figure 2.11 A kinetic study demonstrating the importance of GST in quantifying GSH
in erythrocytes. The change of fluorescent intensity as a function of time is shown for
three GST concentrations (0.5-2.5 U/ul).

63

continuous flow measurements. The continuous flow method will be able to monitor the
redox status of glutathione at a rate that is limited by the data acquisition frequency. Also
this assay is amenable to microchip studies and continuous flow studies that are
important in evaluating the redox status of RBCs in disease status.

Such a dynamic measurement scheme is important in when attempting to measure
the effects of oxidant stressors on RBCs, especially considering that patients with type 2
diabetes have been reported to have lower levels of GSH in their RBCs in comparison to
healthy controls.4 Moreover, recently, it has been shown that oxidative stress has a direct
effect on the ability of erythrocytes to release NO stimulating ATP.4 In fact, the trends in
the ability of erythrocytes to release ATP upon mechanical deformation were similar to

the ability of GSH to be remained in its antioxidant form or reduced form of GSH.4

2.7 Conclusions

GSH, the most abundant non-enzymatic antioxidant in RBCs, has been
determined in both static and flow systems. Although GSH has been determined using
numerous techniques, the work presented here represents the first quantitative
determination of GSH using a bimane probe without any prior separation step.4
However, cells are dynamic and homeostatic systems and will attempt to return to their
pre-oxidative attack conditions. Therefore, it is imperative that the measurement of GSH
oxidation to the GSSG dimer occur in near real time with the method that is fast and
simple.4 This type of monitoring would not be possible with any type of system that

required a discrete sampling or injection like HPLC or CE.4 Moreover, this assay system

64

was employed in establishing the importance of antioxidant status of erythrocytes in the

ability of the erythrocytes to release ATP, a known nitric oxide stimulus.

65

References

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Newton, G. L.; Dorian, R.; Fahey, R. C., Analysis of biological thiols -
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1995; (251), 123-133.

70

CHAPTER 3

Modulation of erythrocyte derived ATP release in response to hydroxyurea

and nitric oxide

3.1 Hydroxyurea on vasculature

Sickle cell disease (SCD), an autosomal recessive disorder, is one of the most
prevalent genetic disorders in the world.l It was the first genetic disease to be identified at
the molecular level, with the single amino acid mutation of glulamate to valine in the [3
sub unit of hemoglobin resulting in the formation of a new hemoglobin molecule called
hemoglobin S (HbS). This abnormal HbS alters the quaternary structure of hemoglobin in
deoxygenated conditions resulting in the sickle shaped formation of the RBCs. Sickling
of RBCs is responsible for the majority of the pathogenic events that result in the clinical
aspects of the disease.

Even though it was the first disease to be identified at the molecular level in 1949,
until the 19903 there was essentially no therapy for improving the complications
associated with the disease. In 1995, Charache et al and Schechter et al showed the
possibility of using hydroxyurea as a new therapeutic agent in the management of SCD.2'
4 Since then, hydroxyurea has been used as a clinically proven therapy for SCD in variety
of situations.5

Hydroxyurea, a hydroxylated derivative of urea, is an inhibitor of the

ribonucleotide reductase enzyme. It is water soluble and also easily absorbs into the

71

NH2 IFIH
OH

Figure 3.1 Structure of hydroxyurea

body and hence therapeutically easy to administer. The prime rationale of the beneficiary
effects of hydroxyurea in SCD is its ability to induce fetal hemoglobin (HbF) levels in
RBcs.6

The increase in HbF alters the kinetics and thermodynamics of HbS
polymerization, thereby reducing the probability of sickling of the RBCs in deoxygenated
conditions.7 Several studies have suggested the possible mechanism of the augmentation
of HbF upon hydroxyurea treatment. However, the exact mechanism of action still needs
to be elucidated. Nevertheless, hydroxyurea appears to have many other beneficial effects
in SCD patients through other possible mechanisms. The main evidence for this
explanation comes from the clinical studies carried out in patients undergoing
hydroxyurea treatment. Specifically, the main characteristics of SCD are painful
vasoocclusive crisis, acute chest syndrome, and hemolytic anemia. Interestingly, many
clinical improvements have been observed in patients undergoing hydroxyurea treatment

even before any detectable rise in HbF in the RBC. Conversely, clinical improvements

72

have been observed upon hydroxyurea treatment have diminished upon discontinuing the
treatment even though HbF levels are significant in the blood stream. These observations
suggest other possible pathways of action for hydroxyurea in SCD.

Membrane damage is often associated with the sickling process and the damaged
membrane is thought to increase adherence of RBCs to the endothelial cells and damage
the endothelium.3 Abnormal interactions between sickled RBCs and endothelial cells are
the primary factors in instigating the microvascular occlusions in SCD, leading to painful
vasoocclusive crisis.8 One of the important benefits of hydroxyurea treatment in SCD,
other than the HbF increase, is the reduction in red cell- endothelial cell interaction.
Erythrocytes from hydroxyurea treated patients have a reduced adhesion to the
endothelium and reduced expression of cytokines and other adhesion molecules.

Sickled RBCs are more susceptible to lysis; hence hemolytic anemia cell lysis is a
widespread symptom of SCD. Apart from that, these cells are less deformable than those
obtained from healthy controls and contribute to the abnormalities in blood flow. These
factors indeed contribute to the major pathophysiological complications associated with
the disease. Chronic hemolytic anemia, vasoocclusive crisis and acute chest syndrome are
among the wide spectrum of clinical manifestations of the disease and all these symptoms
share the rheological abnormalities of the vascular system. As a result, SCD is considered
a disease of the circulatory system, even in the presence of its genetic basis.
Interestingly, hydroxyurea is known to improve rheological characteristics such as
increased red cell deformability, improved RBC hydration and reduced red cell density.9
Zoumbos et al have demonstrated the improved deformability of red blood cell

membrane upon treatment with hydroxyurea.lo In contrast, an in vitro study performed by

73

Kim-Sharpio et al have demonstrated reduced RBC deformability at very high
concentrations of hydroxyurea.H

Reduced NO bioavailability in SCD is an important component of the
abnormalities associated with vascular function.’2 Intracellular HbS polymerization
destabilizes the RBC membrane, resulting in RBC lysis and increased cell free
hemoglobin content in the plasma. Decreased levels of NO in the vasculature
result from the fast reaction of NO with cell free hemoglobin and reactive oxygen
species.1 Hydroxyurea has an effect on increasing the bioavailability of NO in SCD. It is
known to exert some of its effects via the formation of NO.'3"5 Patients who undergo
hydroxyurea treatment have higher levels of NO metabolites in the blood stream,
suggesting the possibility of NO formation by hydroxyurea. However, the mechanism of

NO formation by hydroxyurea is still not clear and requires further investigation

3.2 Nitric Oxide on vasculature

Nitric Oxide (NO) has been recognized as an ubiquitous intra and extra cellular
signaling molecule in diverse pathophysiological processes such as cardiovascular
homeostasis, neurotransmission and non specific immune responses. This small, diatomic
free radical is considered as one of the most important signaling molecules in the
cardiovascular system. It is well established that NO is involved in vascular homeostasis
by regulating the vascular resistance, smooth muscle relaxation and inhibiting RBC and
platelet activation, aggregation and adhesion in the circulatory system.”17

NO is synthesized in the vascular endothelium in response to physiological

stimuli and contributes to ~ 20-30% of the basal human blood flow.17 NO synthesized in

74

endothelial cells can be released both abluminally and luminally.l8 When released
abluminally, NO can interact with the vascular smooth muscle cells underlying the
endothelium, stimulating vascular relaxation, and thereby resulting in an increase in
vascular caliber. NO released luminally, which is released into the vessel, can interact
directly with the components of the blood and thereby contribute to several physiological
events. 1 8‘ l 9

NO is produced enzymatically by the nitric oxide synthase (NOS) protein and
requires five cofactors; flavin adenine dinucleotide (FAD), flavin mononucleotide
(FMN), tetrahydrobiopterin (H4B), nicotinamide adenine dinucleotide phosphate
(NADPH) and a heme group for its full activity.20 Among the three NOS isoforms
present in mammalian cells, endothelial NOS (eNOS) and neuronal NOS (nNOS) require
activation by the calcium - calmodulin complex. In the endothelium, NO is generated
during the conversion of L- ariginine to L-citrulline by NOS upon activation by the
calcium - calmodulin complex. Synthesis of NO by endothelial cells is a tightly regulated
process and significantly contributes to regulation of the vascular caliber.

In addition to the production of NO by endothelial cells, human RBCs do contain
both inducible and constitutive forms of NO synthase genes, and thus have the capability
of producing their own NO and modulating several functions?"22 Therefore, it has been
suggested that RBC derived NO has a contributory role in regulating the physiological
behavior of the RBC as well as contributing to the intravascular NO pool. Therefore, NO

can be considered as an intra- and extracellular signaling molecule that mediates several

physiological functions in regulating the vasculature.

75

The bioavailability of NO in the vasculature depends on the NO diffusing from
the endothelium and NO produced by other cells. However, the high reactivity between
hemoglobin and NO was determined to be the reaction that resulted in a decreased
bioactivity of NO in the vascular system. In addition, several studies have shown the
encapsulation of hemoglobin in RBCs and the RBC free zone near the vessel wall have
dramatically reduced the consumption of NO by hemoglobin compared to the reaction
rate of cell free hemoglobin with NO and thereby provide evidence for the preservation
of bioactivity of NO in the vasculature.23’24 It has been experimentally shown that NO
reaction with RBC encapsulated hemoglobin is ~1000 times slower than the reaction with
cell free hemoglobin.

Apart from the diffusion barrier model of NO preservation in the cardiovascular
system, there has been another mechanism that explains the preservation of NO in the
vasculature. The formation of nitrosylhemoglobin [HbFe(II)NO] and S-
Nitrosohemoglobin (SNOHb) in RBCs is of current interest, suggesting that NO is the
third molecule transported by hemoglobin in the vasculature.”26 However, while this
conservation of NO by reaction with hemoglobin is still attractive, it is a controversial
concept. According to the SNOHb model, NO reacts first with the deoxygenated
hemoglobin in oxygenated RBCs forming [HbFe(II)NO], which as has no vasodilator
activity. NO is then transferred to cystein -98 residue of the [3 subunit of hemoglobin
upon oxygenation to form SNOHb. NO can be released by SNOHb under deoxygenated
conditions in an allosteric mechanism, thereby preserving the NO bioavailability in the
vasculature.”27 SNOHb is stable in oxygenated RBCs, but easily releases NO in

deoxygenated environments, thereby resulting in its RBCs vasodilator potency.28 The

76

release of NO operates through the anion exchanger protein AEl at the RBC
membrane/cytosol interface.

Despite the controversies of NO reactivity preservation in the vasculature, it is
well established that NO plays a central role in the cardiovascular system. Impaired
release of NO by RBCs and endothelial dysfunction can limit the bioavailability of NO in
the vascular system and is known to have direct implications in several disease
conditions. Impaired release of NO by the RBC or dysfunction of RBCs NO synthesis by
the RBC have been associated with the pathogenesis of hematologic, thrombotic, and
ischemic disorders.26 More specifically, RBC—NO processing impairment has been

3"” and heart failure.34

associated with diabetes,29 SCD,30 pulmonary hypertension,
Detailed experimentation of disease conditions have suggested the possibility of impaired
vasodilation by RBC derived NO or different amounts of SNOHb in RBCs. It is evident
from studies that irregulation of blood flow may originate at different stages, and the
concentrations of the NO, or the bioavailability of that NO, result in pathological
complications.26 Finally, it is now established that RBCs provide a novel vasodilator

activity by producing NO via the NOS pathway and preserving NO via SNOHb

formation.

3.3 Effect of hydroxyurea and NO on erythrocyte derived ATP release

It has been reported that ATP released from RBCs in response to several

physiological stimuli such as mechanical deformation and hypoxia is a stimulus for

endothelial derived NO synthesis.35’36 The released ATP from RBCs can bind to the P2Y

77

receptors on the vascular endothelial cells, thereby stimulating the production of NO in
the endothelial cells. The produced NO can diffuse in to the underlying smooth muscle
layer where it can participate in the regulation of vascular caliber. Therefore, RBC
derived ATP is considered as a determinant of regulating vascular resistance.37

RBC deformability plays a major role in proper functioning of the RBCs in
relation to efficient oxygen and carbon dioxide distribution in the microvasculature. The
ability of RBCs to release ATP in response to mechanical deformation and its importance
in vascular resistance have led to the exploration of small molecules, metabolites or drugs
that alter the deformability of RBCs. These molecules may open up a new field of current
medical research for potential therapeutic applications in rheological abnormalities
associated with disease conditions.

Hydroxyurea is the currently the only FDA approved treatment for patients with
sickle cell anemia. The improvements of rheological properties such as increased cellular
deformability and improved blood flow have been observed in patients undergoing
hydroxyurea treatment. There are also several studies associated with RBC deformability
and hydroxyurea. This relationship between hydroxyurea and RBC deformability has
inspired us to investigate the effect hydroxyurea on RBC derived ATP release. However,
there is no exact mechanism to demonstrate the ability of hydroxyurea in regulating
blood flow or rheological properties in sickle cell patients. NO is another important
molecule and is recognized as a potent endogenous vasodilator. Several researches have
suggested that NO modulates RBCn deformability?!" 3840 Korbut et al have shown that
NOS synthase inhibitors such as N-nitro-L-arginine methyl ester (LNAME) have an

effect on erythrocyte deformability in septic shock.” 4'

78

In addition, it has been shown by the same authors that RBC deformability was
influenced by polymorphonuclear leucocytes through a mechanism involved in NO
release.39 Furthermore, NO donors such as spermine NONOate have been shown to be
involved in increased deformability of RBCs. In contrast, there has been a study showing
reduced RBC deformability in response to NO.42 These contradictory findings have taken
attention away from the potential significance of NO in the microcirculation. Moreover,
these inconsistencies led us to explore more closely the effect of NO in RBC mechanical
properties. Also, the relationship of hydroxyurea and NO in RBC deformability provided
new insights and inspired studies to examine the effect of these small molecules on RBC
derived ATP release.As both hydroxyurea and NO have the ability to modulate RBC
deformability, it is possible to hypothesize that hydroxyurea and NO can modulate the
RBC derived ATP release and there by contribute to the rheological properties of

erythrocytes.

3.4 Chemiluminescence measurement of deformation induced ATP release from

RBC

Chemiluminescence is the emission of electromagnetic radiation as a result of a
chemical reaction. When the light emitting reaction is arising from a living organism, it is
commonly known as bioluminescence. Fire flies (Photinus pyralis) emit light by the
reaction of luciferin and molecular oxygen. The rate of this reaction is very slow, thus
luciferase, a bioluminescence producing enzyme, in fire flies catalyzes the reaction with
the use of ATP. This enzymatic reaction is specific for ATP and has high quantum

efficiency. Therefore, luciferin/luciferase reaction with ATP can be employed to

79

quantitatively determine reactions in biological samples. As shown in figure 3.1 luciferin
and ATP combine at the active site of the luciferase forming luciferyl-AMP intermediate.
Molecular oxygen reacts with the luciferyl-AMP forming an excited luciferyl -AMP*
state. Upon relaxing, the excited intermediate emits a visible range light (green glow).
The chemiluminescence intensity is proportional to the amount of luciferin molecules
excited.

The attractiveness of chemiluminescence as a detection technique in bioanalytical
applications is its simplicity. The reaction does not require any light source other than the
light emitting reaction, making chemiluminescence a simple technique with no
interference from an excitation source with high sensitivity and good limits of detection.
The simple instrumentation used to detect the emitted light makes the assay robust and
easy to use. A photomultiplier tube (PMT) is the most widely used detector in
chemiluminescence. Good sensitivity, broad dynamic range and applicability to a wide
range of reactions have made the PMT a traditional chemiluminescence detector. The use
of chemiluminescence, in concert with a model that mimics the RBC circulation in the
arterioles and capillaries, has been employed to determine the ATP release from RBCs in
response to mechanical deformation.43"45 Continuous pumping of RBCs through silica
microbore tubing is close to the physiological conditions, thereby exposing RBCs to a
mechanical stress that would be similar in the circulation. This novel microflow
technique has advantages over the previously employed filtration method used by
Sprague et al to quantify RBC derived ATP release. The filtration technique employs
vacuum induced flow of RBCs through a membrane filter to place mechanical stress on

RBCs. The release of ATP through filtration is detected using the same ATP assay, but in

80

an “off line” manner. This may lower the detection limits and lower the sample
throughput.44

In contrast, the method employed in this study was able to monitor the
chemiluminescence intensity in near real time over a longer period of time. This is
advantageous in monitoring the real time changes in ATP release from RBCs in response
to physiological stimuli. Also this method enabled a more clear understanding of the
parameters that affect RBC derived ATP release in vivo as it mimics the internal
circulation in a in vitro platform that allow understanding of the change of rheological

properties on RBC derived ATP release.46

81

HO

8 N 002-
N S
Mg2+
O
O
HO 3 /N O—g—O O Adenine
/: , (I) m}? PPi
N S
l 02 HO OH
O'
/ O
O O
HO 3 /N 0 [fl 0 O Adenine
/> < I-
N s 0
HO OH

8 N O
/ .
/ +C02+AMP+H

 

 

S /N O L' ht
> < + 19
/ f
N 8

Figure 3.2 Schematic mechanism of the chemiluminescence generation from luciferin
and luciferase in the presence of ATP. Note the importance of molecular oxygen in the
generation of the intermediate of excited oxyluciferin.

82

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3.5 Experimental

3.5.1 Methods and materials

Generation of washed red blood cells

Male, New Zealand white rabbits were anesthetized with ketamine (8 ml/kg, i.m.) and
xylazine (1 mg/kg, i.m.) followed by pentobarbital sodium (15 mg/kg i.v.). A cannula
was placed in the trachea and the animal was ventilated with room air. A catheter was
placed into a carotid artery for administration of heparin and for phlebotomy. After
heparin (500 units, i.v.), animals were exsanguinated. Human blood was obtained by
venipuncture without the use of a tourniquet (antecubital fossa) and collected into a
heparinized syringe. Blood was centrifuged at 500 x g at 4°C for 1.0 min. The plasma and
buffy coat was discarded. RBCs were resuspended and washed x 3 in a physiological salt

solution [PSS; in mM, 4.7 KCl, 2.0 CaClz, 140.5 NaCl 12 MgSO4, 21.0

tris(hydroxymethyl)aminomethane, 11.1 dextrose with 5% bovine serum albumin (final

pH 7.4)]. Cells were prepared on the day of use.

Preparation of A TP standards

All the reagents were purchased from Sigma chemical company, St. Louis, MO
unless otherwise noted. A 100 uM stock solution of ATP was prepared by adding 0.05 51
g of ATP to 1000 m1 of distilled and deionized water (DDW). The standard solutions (0,
25, 50, 100, 150 nM) were made by adding 0, 67.5, 125.0, 187.5, 250.0, and 375.0 111 of

100 11M ATP stock solution to PSS, respectively. All the standards were prepared in 25

84

ml volumetric flasks on the day of use. PSS was prepared by first combining 25 ml of
Tris buffer and 25 ml of Ringer’s buffer and then adding 0.50 g of D-Glucose and 2.50 g
of bovine albumin fraction V (fatty acid free). The entire solution was then diluted to 500
ml by adding DDW and adjusted the pH to 7.35-7.45. The PSS solution was filtered three

times using a 0.45 pm filter (Corning, Fisher Scientific) before use.

Preparation of Luciferin-Lucijerase reaction mixture

Firefly extract containing luciferin and luciferase was dissolved in 5.0 ml of
DDW; an additional 0.002 g of synthetic luciferin was added to the firefly mixture to
enhance the sensitivity. Chemiluminescence intensity generated by ATP and luciferin-

luciferase reaction was measured with a PMT.

Other Reagents

 

Spermine NONOate: Spermine NONOate solution was prepared by dissolving 1.0 mg of
spermine NONOate (Cayman Chemicals, Ann Arbor, MI) in 1 ml of 0.20 M NaOH. The
concentration of the resulting spermine NONOate solution is 38 mM. The ranges of
concentrations were prepared by adding different volumes of the stock solution in

deoxygenated PBS 7.4 buffer.

Hydroxyurea: For studies involving hydroxyurea, a 4 mM hydroxyurea solution was

 

prepared by dissolving 0.004 g of hydroxyurea (Sigma Aldrich, St. Louis, MO) in 25.0
ml of PSS. The range of concentrations (0 — 200 11M) were made by diluting appropriate

volumes (0 — 500 pl) of the stock solution in a solution of 7% RBCs made in PSS. When

85

preparing the hydroxyurea treated RBCs, hydroxyurea was first added to PSS before
adding RBCs to create a homogenously treated RBC solution. RBC samples were then
incubated for 20 minutes at room temperature before the measurement portion of the

analysis.

Diamide: For studies involving diamide, a 2 mM stock solution was prepared by
dissolving 0.0344 g of diamide in 100 ml of DDW. The final concentration of 20 11M

diamide was made by adding 1.0 ml of2 mM diamide in 9.0 ml of PSS.

3.5.2 Instrumentation

Chemiluminescence measurement of deformation induced RBC derived A TP release

All RBC solutions were diluted to a hematocrit of 7% in PSS. To measure the
RBC derived ATP release, the luciferin- luciferase reaction mixture was placed in a 500
pl syringe (Hamilton, Fischer Scientific). The RBC solution was placed in another 500 111
syringe and both syringes were loaded on to a syringe pump (Harvard Apparatus, Boston,
MA) and connected to two 25 cm sections of silica microbore tubing having an internal
diameter of 50 um and an outer diameter of 362 um (Polymicro Technologies, Phoenix,
AZ). The microbore tubes were then connected to a T-junction (U pchurch Scientific, Oak
Harbor, WA) having a volume of 256 nl. The combined contents were passed through a 6
cm section of microbore tubing having an internal diameter of 75 um. The polyimide

coating was removed from the tubing and placed on the PMT. The resultant

86

chemiluminescence produced by the RBC derived ATP and the luciferin-luciferase

reaction was detected by a PMT housed in a light excluding box (figure 3.3).

3.6 Results and Discussion

It is well known that RBCs release ATP in response to mechanical deformation. It
has also been reported that the amount of ATP released from RBCs is proportional to the
overall RBC deformability. The previous works by other groups have also demonstrated
the effect of hydroxyurea on RBC deformability. However, there was no study that
determines the ability of hydroxyurea to release ATP from RBCs. In order to investigate
the effects of hydroxyurea on RBC derived ATP release, as well as the cellular
deformability, a 7% solution of RBCs was incubated in PSS containing 50 11M
hydroxyurea for 20 minutes. The resultant ATP release from RBCs was measured using
the microflow technique described. As expected, an increased release of ATP was
observed for 7% RBCs incubated with 50 uM hydroxyurea compared to the control 7%
RBCs (figure 3.4).

This primary data obtained from figure 3.3 demonstrates that deformation induced
ATP release from RBCs is increased when subjected to hydroxyurea treatment. RBCs
typically contain millimolar levels of ATP in the cytosol. However, the above data does

not eliminate the possibility that the released ATP is not due to RBC lysis.

87

 

.0
co

1 L

.0
\l

 

 

O
C)
.1144 L4 1_1_1

.0
or

 

0.3 3

ATP release (11M)
0
A

0.2 5

0.1 5

 

l

 

 

 

 

 

l

7% RBC 7% RBC + 50 1.1M HU

 

0.0 '

Figure 3.4 Quantitative determination of ATP release from RBCs in the presence of
hydroxyurea using flow through chemiluminescence detection. The black bar represents
the ATP release from a 7% hematoctrit of RBCs while the gray bar represents the ATP
release from a 7% hematoctrit of RBCs incubated with 50 uM hydroxyurea for 20
minutes. The two bars are significantly different ((P< 0.03) and error bars are reported as

SEM for n = 8 rabbits.

88

Therefore, another experiment was designed to provide evidence that the
increased ATP release was not due to RBC lysis. Glbenclamide, a known inhibitor of the
CFTR protein, was employed to inhibit the signal transduction pathway of ATP release
from RBCs. As CFTR is a required component of the pathway, the inhibition of the
protein results in decreased ATP release. Figure 3.5 shows decreased ATP release from
RBCs incubated with glybenclamide followed by hydroxyurea treatment compared to the
RBCs incubated with hydroxyurea alone. A slight increase of ATP release from RBCs,
compared to untreated RBCs, could be a possibility of partial inhibition of CFTR protein
or the insufficient incubation time of RBCs and glybenclamide. If the cells have been
lysed by the hydroxyurea treatment, one would still expected to see an increase in ATP
release from RBCs incubated with 100 uM glybenclamide. The results from this study
(shown in figure 3.5) confirm that the effect of hydroxyurea on RBC derived ATP release
is not due to cell lysis.

The above experiments did not verify that the ATP release was deformation
induced. Thus, another experiment was carried out to distinguish if the effect from
hydroxyurea was deformation induced. Similar experimental conditions to those
described above were followed for a non flow based measurement where the ATP release
was determined after incubating the 7% RBC solution for 20 minutes in hydroxyurea
without subjecting the RBCs to flow. Interestingly, hydroxyurea did not increase as much
as RBC derived ATP release in the non flow system, compared to the flow based
experiments suggesting the ability of hydroxyurea to modulate the RBC deformability

(figure 3.7).

89

 

0.8 .

0.7 {

 

0.6 {
0.5 a
0.4 -

0.3 ~

ATP release (nM)

0.2 1

0.1 -‘

  

 

 

 

 

 

 

 

 

0.0 r
7% RBC 7% RBC + HUT), RBC + GW + HU
0

Figure 3.5 The effect of glybenclamide on ATP release from RBCs in the presence of
hydroxyurea. The gray bar represents the ATP release from a 7% hematoctrit of RBCs
incubated with 50 uM hydroxyurea while the dark gray bar represents the ATP release
from a 7% hematoctrit of RBCs incubated with 100 uM glybenclamide followed by 50
uM hydroxyurea. The value of (*) is significantly different from others (P<0.01) and the
error bars are reported as SEM for n = 5 rabbits.

90

2.0

 

1111141
‘-

 

1.6fi

1.4 .

**

1.2 -

 

 

 

1.0-j
0.8 «j

0.6 5

Normalized ATP release

0.4 j

0.2 3

 

 

 

 

 

 

 

 

 

 

00 I I 35314351?
with flow non flow

Figure 3.6 The increased ability of hydroxyurea to release ATP from RBCs under
flow conditions. The black bars represent the ATP release from a 7% hematoctrit of
RBCs while the gray bars represent the ATP release from a 7% hematoctrit of RBCs
incubated with 50 uM hdroxyurea for 20 minutes. The bars corresponding to flow
conditions (*) are significantly different (P<0.001). However, the bars denoted as (**) are
different at P<0.05. Error bars were reported as SEM for n = 4 rabbits.

91

Encouraging results were obtained for hydroxyurea treated RBCs in vitro;
therefore a series of experiments were carried out to determine the hydroxyurea
concentration that resulted in the highest ATP release. The data in figure 3.7 and 3.8
show the effect of hydroxyurea concentration on RBC derived ATP release. RBC derived
ATP release increased up to a concentration of 100 uM hydroxyurea in 7% RBCs.
However, as shown, the release of ATP decreases, eventually reaching a value equivalent
to the 7% RBC solution without hydroxyurea. This data was initially confusing, but
repeated experiments indeed conferred the same observations. These data suggested the
possibility of the NO forming ability of hydroxyurea, as NO has been shown to modulate
the erythrocyte deformability. Many studies have indicated the ability of hydroxyurea to
produce NO, but the mechanism of NO producing reactions by hydroxyurea is still not
clear and somewhat controversial. However, the similarity between hydroxyurea and NO
on cellular deformability led to a more detailed examination of the effect of NO on RBC
derived release of ATP.

Initially, a 7% solution of rabbit RBCs were incubated with 100 nM spermine
NONOate for 10 min followed by the quantitative determination of ATP. An increase in
ATP release was observed for the 7% RBCs incubated with spermine NONOate
compared to the control 7% rabbit RBCs incubated for 10 minutes in the absence of
NONOate. (figure 3.9). The principle rationale behind this experiment was NO’s ability
to modulate the RBC deformability. Also, as stated above, the NO producing ability of
hydroxyurea was taken into consideration as many proposed mechanisms were proposed

for NO generation by hydroxyurea

92

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n b h p P n p F P b — p n n h — p n h n b b n u h n n P n b n b p n p - PL P b F L L Fir b h p p n _ @010
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93

 

2.4
2.2
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4

Normalized ATP release

0.2
0.0

 

0 c \3 \5 0
1 | “a “a “a

1°l°

Figure 3.8 Effect of hydroxyurea concentration on deformation induced ATP release
from RBCs. Highest ATP release was observed for 7% hematoctrit of RBCs incubated
with 100 uM hydroxyurea for 20 minutes. After that, the ATP release started decreasing
reaching to a value that was not significantly different from untreated RBCs at 200 M
hydroxyurea. The error bars are reported as SEM for n =7 rabbits.

94

 

 

0.8 f

0.6 {

Normalized ATP release

0.4 f

0.2 —j

 

 

 

 

 

0.0 _‘ ._ . i
7% RBC 7% RBC + 100 nM NO

 

Figure 3.9 Quantitative measurement of deformation induced ATP release from 7%
RBCs in the presence and absence of NO donor, spermine NONoate. The black bar
represents the ATP release for untreated hematocrit of 7% RBC, while the gray bar
represents the 100 nM NONOate treated hematoctrit of 7% RBCs. n=5 and the error or
bars are reported as SEM.

95

The results obtained during the preliminary studies led to further experimentation
involving NO and ATP release with different spermine NONOate concentrations. A
series of 7% RBC samples were made using varying spermine NONOate concentrations
(0 — 1000 nM) and incubated for 10 minutes. Deformation induced ATP released was
measured using the luciferin- luciferase reaction. As shown in figure 3.10 and 3.11 the
amount of ATP release from the RBCs was concentration dependant and the highest
amount of ATP was released for the sample containing 250 nM spermine NONOate.
Subsequently, the ATP release from RBCs started decreasing; reaching a value that was
less than the normal release of ATP from 7% RBCs.

These data showed the same trend as in figure 3.8 The initial increase of ATP
release may be due to the interaction of NO with cystein residues in hemoglobin
(formation of SNOHb) leading to a conformational change of the hemoglobin. This could
lead in deformational changes in the RBC membrane. Conversely, the decreased release
of ATP from RBCs at higher spermine NONOate concentrations may be due to the
higher oxidative stress caused by reactive nitrogen species in RBCs. Higher oxidant
stress could damage the membrane properties; especially the oxidation of spectrin, which
can reduce the RBC deformability.

The change in deformability was observed indirectly by measuring ATP release
from RBCs. Therefore, diamide, a known oxidant, was used to oxidize cell membrane
proteins such as spectrin. Upon oxidation, spectrin forms dimers making the cell
membrane less deformable. The ATP release from RBCs is proportional to cell

deformability; therefore, the same technique described previously can be

96

 

 

 

 

 

 

 

 

 

 

0.075—
0.0703
1
fi 0.065;.H..lll.l_ ,
z. : {J H
.2 . c
93 0.060:
.9. «
8 1 ll l l l ll
c 0.0554 , , , . d
a, __ , _
o . fl
‘0 .
5 ‘Jllll 'l l Ml W J"1..
E 0.050‘1 b
2 .
E 1
2 0.0453
0 * l l
0040-: "ll NIH | H H "H a
0.035%....“M.,..-fh........,-.......fi...............
0 2 4 6 a 10 12 14 16 1a 20
Time(sec)

Figure 3.10 Chemiluminescence intensity change for RBC samples treated with
varying concentrations of spermine NONOate. Spectrum (a) is the chemiluminesence
intensity recorded for 7% hematoctrit of RBCs reacted with luciferin-luciferase mix in
the flow system while spectra b, c, d, and e represent the 7% hematoctrit of RBCs
incubated with 100, 250, 500 nM spermine NONOate, respectively. The signal intensities
were taken at a rate of 10 points/sec for 20 seconds time period.

97

 

0.5

 

0.4 - ll

0.3 1

ATP release (nM)

0.1 -

 

 

 

0.0 -
NO

° 0
7 / 100 “M $73.» 250 “M “0 +500 “M 1o/..+7 50 nM7'3/31000 “M NO

7 °/o + 7 0/0

Figure 3.11 Effect of spermine NONOate on deformation induced ATP release from
RBCs. The ATP release was initially increased gradually with increasing concentration of
NO donor and the highest release was observed for 7% RBCs incubated with 250 nM
Spermine NONOate. Then, the ATP release started decreasing gradually reaching to a
point at 1000 nM where the ATP release was not significantly different from the initial
untreated 7% RBCs. The two bars represented as (*) are significantly different in P< 0.01
and the two bars represented as (***) are significantly different in P<0.05. n = 4 rabbits
and the error bars are reported as SEM

98

employed to investigate the effect of NO on diamide treated RBCs. The effect of NO on
RBCs derived ATP release was not prominent when the cells were first incubated with 20
uM diamide followed by 100 nM spermine NONOate (figure 3.12).The
chemiluminescence intensity was measured after a 10 minute incubation with the NO

In conclusion, both hydroxyurea and NO were shown to have an effect on
modulating the RBC derived ATP release. Also, both molecules resulted in the release of
different amounts of ATP from RBCs in response to mechanical deformation, and most
interestingly, correlated perfectly. The increased ATP release was observed up to a
certain concentration and then started decreasing. This study clearly demonstrates the
relationship between hydroxyurea and NO on erythrocyte derived ATP release. However,
the data do not provide evidence for a proper mechanism of action of hydroxyurea on
ATP release. It suggests the ability of hydroxyurea to change the overall RBC
deformability in a concentration dependant manner.

Hydroxyurea is a proven therapy for SCD because it helps in the reduction in the
number of vasoocclusive crisis, hospitalization and the rate of blood transfusions. More
over, it reduces the chances of having acute chest syndrome, the most dangerous
complication of SCD. All these improved clinical complications could be related to
improved blood flow. As ATP is a determinant in endothelial derived NO production and
vasodilatation, the ability of hydroxyurea to release ATP from RBCs could be another
mechanism of action of hydroxyurea in SCD patients. To date, it has been shown that
hydroxyurea can improve the cellular deformability, hydration status and reduce the

RBC- endothelial interactions. This study may be another attempt of

99

 

 

 

Normalized ATP release
N
n 1

W“

\\

 

 

 

 

 

s\\\\\\\\\\& ,1

 

   

//

7% R305 7% RBCs + 7% + diamide 7% RBCs + diamide
hydroxyurea + hydroxyurea

 

 

 

Figure 3.12 The effect of cellular oxidant, diamide on RBC ATP release in the
presence of spermine NONOate. The black bar represents the ATP release for hematocrit
of 7% RBCs while gray bar with medium lines represents the ATP release for 7% RBCs
in 250 nM spermine NONoate. Obviously, the lowest ATP release was found to the 7%
RBCs incubated with 20 uM diamide (dark gray bar). However, the ATP release is not
increased significantly for 7% RBC incubated first with 20 uM diamide followed by 250
nM spermine NONoate. The (*) and (**) are significantly different in P<0.001 for n =3
rabbits and the error bars are reported as SEM.

100

demonstrating a possible relationship of hydroxyurea in relation to the improved blood
flow in sickle cell patients undergoing hydroxyurea treatment. Further experimentation is
necessary to conclude this novel mechanism of action described by the effect of

hydroxyurea on erythrocyte ATP release.

3.7 Conclusions

It is well accepted that RBCs release ATP in response to mechanical deformation.
There have also been reports demonstrating the effect of hydroxyurea on RBC
deformability. However, this is the first study that demonstrates the effect of hydroxyurea
on ATP release from RBCs. The effect is only seen in the flow system, where RBCs are
subjected to continuous shear stress, suggesting a contributory role of hydroxyurea on
RBC membrane properties. The modulation of ATP release from RBCs is dependant on
varying hydroxyurea concentration. Moreover, the NO donor compound, spermine
NONOate gave the same trend as hydroxyurea, suggesting a mechanism of NO formation
by hydroxyurea within RBCs. The formed NO is thought to responsible of the observed

ATP release from RBCs, as NO is known mediator of RBC deformability.

101

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Califf, R. M.; Singel, D. J.; Piantadosi, C. A.; Tapson, V. F.; Stamler, J. S., A
Nitric oxide processing defect of red blood cells created by hypoxia: deficiency of
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Academy of Sciences of the United States of America 2005, 102, (41), 14801-
14806.

Pawloski, J. R.; Hess, D. T.; Stamler, J. S., Export by red blood cells of nitric
oxide bioactivity. Nature 2001, 409, (6820), 622-626.

Milsom, A. B.; Jones, C. J. H.; Goodfellow, J.; Frenneaux, M. P.; Peters, J. R.;
James, P. E., Abnormal metabolic fate of nitric oxide in type i diabetes mellitus.
Diabetologia 2002, 45, (11), 1515-1522.

Pawloski, J. R.; Hess, D. T.; Stamler, J. S., Impaired vasodilation by red blood
cells in sickle cell disease. Proceedings of the National Academy of Sciences 0f
the United States Of America 2005, 102, (7), 2531-2536.

Foster, M. W.; Pawloski, J. R.; Singel, D. J .; Stamler, J. S., Role of circulating s-
nitrosothiols in control of blood pressure. Hypertension 2004, 45, (1), 15-17.

Foster, M. W.; Mcmahon, T. J.; Stamler, J. S., S-Nitrosylation in health and
disease. Trends in Molecular Medicine 2003, 9, (4), 160-168.

Foster, M. W.; Pawloski, J. R.; Singel, D. J .; Stamler, J. 3., Role of circulating s-
nitrosothiols in control of blood pressure. Hypertension 2005, 45, (1), 15-17.

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V.; Frenneaux, M.; James, P., Red blood cell nitric oxide as an endocrine

vasoregulator - a potential role in congestive heart failure. Circulation 2004, 109,
(11), 1339-1342.

Sprague, R. S.; Ellsworth, M. L.; Stephenson, A. H.; Lonigro, A. J ., ATP: the red
blood cell link to no and local control of the pulmonary circulation. American

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H2722.

Sprague, R. S.; Ellsworth, M. L.; Stephenson, A. H.; Olearczyk, J. J .; Lonigro, A.
J ., Erythrocyte-derived atp produces vasodilation in the rabbit pulmonary
circulation of a nitric oxide-dependent mechanism. Faseb Journal 2002, 16, (4),
A127-A127.

Sprague, R. S.; Olearczyk, J. J.; Spence, D. M.; Stephenson, A. H.; Sprung, R.
W.; Lonigro, A. J ., Extracellular atp signaling in the rabbit lung: erythrocytes as
determinants of vascular resistance. American Journal of Physiology-Heart And
Circulatory Physiology 2003, 285, (2), H693-H700.

Korbut, R.; Gryglewski, R. J., The effect of prostacyclin and nitric oxide on
deformability of red blood cells in septic shock In Rats. Journal of Physiology
And Pharmacology 1996, 47, (4), 591-599.

Korbut, R.; Gryglewski, R. J ., Nitric-oxide from polymorphonuclear leukocytes
modulates red-blood-cell deformability invitro. European Journal of
Pharmacology 1993, 234, (1), 17-22.

Mesquita, R.; Picarra, B.; Saldanha, C.; Silva, J. M. E., Nitric oxide effects on
human erythrocytes structural and functional properties - an in vitro study.
Clinical Hemorheology and Microcirculation 2002, 27, (2), 137-147.

Petros, A.; Bennett, D.; Vallance, F., Effect of nitric-oxide synthase inhibitors on
hypotension in patients with septic shock. Lancet 1991, 338, (8782-3), 1557-
1558.

Bateman, R. M.; Jagger, J. E.; Sharpe, M. D.; Ellsworth, M. L.; Mehta, 8.; Ellis,
C. G., Erythrocyte deformability is a nitric oxide-mediated factor in decreased

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Circulatory Physiology 2001, 280, (6), H2848-H2856.

Edwards, J .; Sprung, R.; Sprague, R.; Spence, D., Chemiluminescence detection
of atp release from red blood cells upon passage through microbore tubing.
Analyst 2001, 126, (8), 1257-1260.

106

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erythrocytes using microbore tubing as a model of resistance vessels in vivo.
Analytical Chemistry 2002, 74, (10), 2274-2278.

Fischer, D. J.; Torrence, N. J.; Sprung, R. J.; Spence, D. M., Determination of
erythrocyte deformability and its correlation to cellular atp release using
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Analyst 2003, 128, (9), 1163-1168.

Carroll, J .; Raththagala, M.; Subasinghe, W.; Baguzis, S.; Oblak, T. D.; Root, P.;
Spence, D., An altered oxidant defense system in red blood cells affects their
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7), 305-311.

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CHAPTER 4

Effect of hydroxyurea on nitric oxide production in erythrocytes: A novel

mechanism for hydroxyurea in sickle cell disease

4.1 Sickle cell disease and nitric oxide bioavailability

The pathophysiology of sickle cell disease has been traditionally characterized by
the polymerization of hemoglobin S (HbS) in erythrocytes under deoxygenated (hypoxic)
conditions resulting in the occlusion of blood vessels.1 The occlusion of blood vessels, or
commonly known as vasoocclusion can contribute to pain crisis, acute chest syndrome,
organ dysfunction and in severe situations, death. In contrast, recent studies have shown
that there are other pathological complications associated with the severity of the disease
in addition to HbS polymerization.2 One such condition is the deficiency of the
endogenous vasodilator, nitric oxide (NO) in the vasculature.3'5

NO, a gas at room temperature, is a small diatomic free radical. The solubility of
NO is 1.9 mM/atm at 25 °C in aqueous solutions, but increases 6-8 fold in non polar
environments such as in cellular membranes.6 The smaller size and the neutrality enable
it to diffuse through cellular structures at a rate of 3300 umz/s".7 NO is also a highly
reactive free radical and its half life depends on the initial concentration and the
composition of the cellular system. Typically, the half life is 1-3 s in physiological

solutions, although several studies have indicated a longer half life in physiological

conditions. The high diffusibility and the short life time have made this molecule suitable

108

as a signaling molecule in various physiological processes. Therefore, NO dependant
vasodilatation is one such important process in vascular homeostasis.

NO is synthesized continuously in the endothelium from L-arginine and is
catalyzed by the eNOS. NO released from the endothelium can diffuse into the
underlying smooth muscle cells and thereby activates smooth muscle relaxation process.
In smooth muscle cells, NO first binds to the heme group of soluble guanylyl cyclase and
triggers the production of cyclic guanylyl mono phosphate (cGMP) which in turn
activates the cGMP dependant protein kinases (PKs). Activation of PKs results in the
decreased levels of the intracellular calcium ion concentrations and this whole process
results in smooth muscle relaxation.8’9 Therefore, NO, produced by the endothelium,
plays a central role in vascular homeostasis by contributing to the control of normal
vascular tone. In addition, NO is involved in reduced platelet aggregation and reduced
RBC adhesion in the vascular system contributing to smoother blood flow.

However, as mentioned above, patients with SCD suffer from reduced NO
bioavailability. Therefore, NO related mechanisms in the vascular system, i.e., regulation
of blood flow, reduced interactions of RBCs with the endotheliumlo’” and inhibition of
platelet aggregation are also affected in patients having SCD. All of these conditions have
been associated with an increased occurrence of several vascular complications such as
vaso occlusive crisis, acute chest syndrome,12 persistent pulmonary hypertension,'3etc,.
Moreover, all of these complications are known to be an effect of reduced bioavailability
of NO in the vascular system and endothelium dysfunction and the increased
consumption of NO by cell free hemoglobin are the known causes of reduced NO

bioavailability.

109

Endothelium dysfunction in SCD is the inability of the endothelium to produce
NO by the NOS pathway.l4 This may be due to low plasma arginine levels or increased
arginase activity in the plasma of SCD patients.15 Arginase is predominant in young
RBCs. Higher production of young RBCs and the higher hemolysis rate of RBCs in SCD
could increase the plasma arginase levels, thereby deplete the arginine reserves in the
plasma of SCD patients. The depletion of arginine may affect the synthesis of NO by the
endothelium as arginine is the substrate for the enzymatic synthesis of NO in the
endothelium.3

Moreover, the consumption of NO by cell free hemoglobin is considered as the
main reaction of decreased levels of NO in SCD patients.4 The rate of hemolysis of RBCs
is high in sickle RBCs due to their abnormal shape, rigidity of the membrane (decreased
deformability) and the increased adhesiveness to the endothelium. In contrast, NO
bioavailability is preserved in the normal vasculature due to the laminar flowing blood
and the compartmentalization of hemoglobin inside RBCs. The cell free hemoglobin
reacts 1000 times faster than the hemoglobin in RBCs. In fact, SCD patients have higher
levels of cell free hemoglobin in the plasma, and thus consume NO rapidly, dramatically
reducing the NO bioavailability. Therefore, the increased hemolytic rate provides the
resistance to NO dependent blood flow in SCD patients.

Both factors described above, i.e. NO scavenging by hemoglobin and the
endothelial dysfunction, have a synergistic effect on NO bioavailability and play an
important role in vascular instability when the patients undergo vascular crisis or acute

chest syndrome where the hemolysis rate is even higher compared to steady state SCD. In

110

addition to vascular instability, patients may suffer pulmonary hypertension, priapism,
stroke, and leg ulcers depending on the location of impaired bioavailability occur.

Considering the factors that affect NO bioavailability in SCD patients, several
therapeutic approaches have been under investigation to restore the NO levels in plasma.
One approach is to directly deliver the NO and NO donor compounds to the circulation.
Inhalation of N0 gas is a FDA approved treatment but requires specialized handling and
administration because of the noxious effects of that gas at higher concentrations. Also,
some studies have shown the use of promising NO pharmaceutical, sodium nitrite as the
NO donor, where the reaction takes place in RBC hemoglobin-derived nitrite reductase
activity. Another possible therapy would be the arginine supplementation where the NO
availability is enhanced by the increased NOS activity."16

In fact, it has been suggested that hydroxyurea, the clinically proven treatment for
SCD, has an additional role as a NO donor in treatment of sickle cell diseasems
However, the mechanism of NO production in vivo by hydroxyurea is not very well
understood and is currently under thorough investigation. However, it is a possibility that
clinical improvements, especially the increased rheological properties observed in

patients that undergo hydroxyurea treatments, are mediated by NO induced vasodilatation

or reduced platelet activation.

4.2 N O forming reactions of hydroxyurea

Primarily, the interests of investigating the possible mechanisms of hydroxyurea

on SCD by means other than induction of HbF levels, arrived from the clinical

111

improvements observed from SCD patients, receiving hydroxyurea before any rise in
HbF levels in the blood. Moreover, the NO forming ability of hydroxyurea has been
proposed as a secondary, but a beneficial effect of hydroxyurea on SCD patients.
However, the exact mechanism of NO formation from hydroxyurea is still not clear and

under investigation.

4.2.1 In vitro NO formation from hydroxyurea

The molecular structure of hydroxyurea, which contains a N-O bond, has led
investigators to focus the attention on researching the NO donor properties of the
hydroxyurea molecule. It is known that the formation of NO from hydroxyurea requires
oxidation of the molecule. Considering this fact, several mechanisms have been proposed
for the formation of NO via oxidation of the hydroxyurea molecule and are shown in
figure 4.1. Pathways 1 and 2 occur through the one electron oxidation of hydroxyurea
forming the nitroxide radical intermediate. Pathway 3 occurs through the formation of
hydroxylamine followed by three electron oxidation producing NO. Supporting the
proposed mechanisms, hydroxyurea has been shown to react with variety of oxidants in
vitro to form nitroxide or the NO free radical. Among them, the reaction of hydroxyurea
with heme proteins, hydrogen peroxide and Cu-Zn-SOD have been thouroughly
studied.19

Several research groups have shown the ability of heme groups to oxidize
hydroxyurea to the NO free radical under in vitro conditions.”23 Stolze et al first

reported the methemoglobin and nitrosylated hemoglobin (HbNO) formation from

112

 

O O
A -1 /"\
NH2 NH :7 NH2 NH ———-> NO + 002 + NH3 ---pathway1
- 1
OH O.
1 2
O O O
NH2 NH——’ -H" NH2 NH '——"NH2 “1 pathway 2
OH O O
1 2 1H20
0
- __, NH + O
No-<—_:fi— HNO+NH2 OH 3 C 2
O
A H o
NH2 NH -2—-r 1"

OH
1

O . . O
heme protein mediated . heme protein mediated
A oxrdatron A - -

 

 

_ oxrdatron
NH2 NH - NH2 NH = N0
OH O.
hydroxyurea nitroxide

Figure 4.1 Three general pathways that depicts the formation of NO from
hydroxyurea under in vivo or in vitro conditions (A). '7 Proposed mechanism of the
formation of NO by hydroxyurea 1n the presence of heme proteins (B).22

113

hydroxyurea.23 Furthermore it was observed that aminocarbonylaminooxyl (HzN-CO-
NHZ) radical and the low spin ferric methemoglobin with hydroxyurea (MetHb-NHOH-
CO-NHZ). Another study by Pacelli et al showed the mechanism of NO formation by
hydroxyurea through oxidation by hemoglobin to form the nitroxide radical intermediate.
The nitroxide radical is further decomposed in vitro to give rise to the NO free radical.22
The several studies confirmed the same results and now it is well accepted that
hydroxyurea reacts with oxyhemoglobin, deoxyhemoglobin and methemoglobin under in
vitro conditions to form HbNO complex, proving the formation of the NO free radical
from hydroxyurea (figure 4.2). Also these studies have confirmed the S-nitrohemoglobin

formation by the reaction of hydroxyurea and methemoglobin.

4.2.2 In vivo NO formation from hydroxyurea

In vivo detection of NO is extremely challenging and difficult due to the rapid reaction of
NO with oxygen and heme proteins. However, several studies have shown the formation
of NO and NO derivatives in the blood stream upon hydroxyurea treatment using
chemiluminescence, electrochemical and electron paramagnetic resonance spectroscopy
methods.”25 Increased levels of plasma NO metabolites such as nitrite, nitrate, nitrosyl
hemoglobin and S-nitrosohemoglobin have been observed with patients undergoing
hydroxyurea treatment providing evidence of a novel mechanism of hydroxyurea. As NO
is a known vasodilator in the vascular system, the clinical efficacy of hydroxyurea on

SCD is known to mediate through regulating the blood pressure and flow.

114

 

 

 

 

 

o o
HbFe (+2)-o2 A L. HbFe(+3) A
oxyHb + ””2 '1'” metHb + NH2 3')“
OH .
o o
HbFe (+2) A , 2 HbFe(+3)._ A
deoxyHb + NH2 if“ metHb ””2 ””2
OH
0 O
HbFe3+ /u\ 4 HbFe? + NH ANH
metHb NH2 3: deoxy Hb 2 (I)
o
A HbFe(+2)
NHZ NH _ NO s HbNO

Figure 4.2 The reactions of HbNO formation by hydroxyurea from the reaction of
oxy, deoxy, and methemoglobin.l7 The reactions of hydroxyurea with oxyhemoglobin
and methemoglobin results in the formation of nitroxide radical which further oxidizes to

form molecular NO.

115

Huang et al reported the high levels of HbNO levels in rats treated with hydroxyurea
suggesting the formation of NO by hydroxyurea metabolism.26 The same group further
tested this observation on human volunteers. Oral administration of hydroxyurea on SCD
patients produced detectable levels of HbNO in the blood stream, but the study did not
confirm mechanism of NO formation via the metabolism of hydroxyurea in vivo.25
However, there have been reports and a consideration that in vivo reactions forming NO
from hydroxyurea with hemoglobin are not sufficient to account for the NO levels
quantified in vivo. The incubation of 10 mM hydroxyurea with whole blood having
oxygenated and deoxygenated hemoglobin did not give detectable levels of HbNO within
a 2 h period.27 These reactions occur at a slow rate, and that they do not demonstrate
support for the increased production of NO metabolites formed upon hydroxyurea
treatment in vivo.20’28 Moreover, the sample calculations and kinetic stidies revealed that
only a small fraction of hemoglobin has reacted with hydroxyurea under physiological
conditions. Therefore, other different pathways that produce NO in vivo may account for
the NO forming reactions by hydroxyurea, and should account for the in vivo production
of NO metabolites from hydroxyurea. The catalase mediated pathway,28 horseradish
peroxidase catalysed pathway29 and urease dependant pathways27 are other sources of NO
forming reactions are the potential in vivo formation of NO from hydroxyurea.
Interestingly, the effect of NO on HbF induction was proposed recently. The
induction of HbF by hydroxyurea is known to mediate through the redox inactivation of

the tyrosyl radical of ribonucleotide reductase enzyme, an effect that can be mediated by

NO and vasodilators. Furthermore, higher levels of hydroxyurea activates cyclic guanylyl

116

mono phosphate (cGMP) production, which in turn activates the protein kinase which
required for HbF production.3 I In addition to functioning as a vasodilator, the induction
of cGMP levels have been observed in patients undergoing hydroxyurea treatment. It is
well known that NO stimulates cGMP production, thereby mediating vasodilatation via
smooth muscle relaxation. Recently, Nahavandi et al, have shown for the first time that
NO derived donor compounds such as hydroxyurea open up a new therapeutic strategy in
NO based therapy for SCD.32

Another important finding in relation to the NO forming ability of hydroxyurea
was the decreased levels of NO metabolites during the vaso-occlusive crisis.
Administration of single doses of hydroxyurea resulted in increase of NO in the plasma
of patients on long term hydroxyurea treatment, and the levels remained above the
physiological values for about a 12 h period. Also, the increased cGMP levels were
observed for a 6 h period for the patients on single hydroxyurea treatment. In summery,
these factors provide evidence for another possible mechanism of hydroxyurea;
specifically, hydroxyurea behaving as a NO donor. Considering the half life of NO in
physiological systems, there is a possibility that hydroxyurea may act as a NO donor as
well as a stimulator for NO production.3 1’33

Moreover, it has been reported that L-arginine, the substrate for NOS, increased
NO production only in the presence of hydroxyurea, implying the use of hydroxyurea as
a NOS stimulus. Furthermore, Cokic et al have shown the induction of eNOS as well as
the stimulation of cGMP levels in endothelial cells upon hydroxyurea treatment.30

However, there is no evidence suggesting that hydroxyurea improves the rheological

properties of erythrocytes via a NOS pathway in erythrocytes.

117

Collectively, the studies carried out in vivo clearly show a measurable increase in
NO metabolites following the administration of hydroxyurea. However, to date, there is
no strong evidence of the in vivo oxidant, or the site of the metabolism of hydroxyurea in

respect to the formation of NO.17

4.3 A novel mechanism of action for hydroxyurea involving erythrocytes

It is now well established that hydroxyurea exerts its beneficial effects in SCD via
a number of additional mechanisms other than the induction of HbF levels. Extensive
research has been conducted over the last decade to identify these mechanisms, and many
research groups have shown reduced red cell- endothelial interactions and improved
rheologial properties of RBCs upon hydroxyurea treatment in SCD patients. Moreover,
SCD is now being considered as a rheological disorder of RBCs, despite the fact that HbS
polymerization due to the genetic mutation causes the major pathogenic events.
Therefore, the investigation of the effects of hydroxyurea on RBC rheological properties
would be beneficial in treating SCD patients in an effective way.
Decreased or loss of membrane deformability is a characteristic feature of sickle
RBCs. This would impair blood flow in small arterioles and capillaries, contributing to
vaso-occlusion and pain crisis. Improved RBC membrane deformability has been
observed with SCD patients undergoing hydroxyurea treatment.34 However, the
mechanism of modulating the RBC deformability by hydroxyurea was not in a priority in
investigations involving to hydroxyurea. The main reason for this may be due to the

controversial findings of erythrocytes deformability in response to different hydroxyurea

118

concentrations. Several studies have shown increased RBC deformability with
hydroxyurea, while other studies have shown decreased RBC deformability upon
hydroxyurea treatment.3 5’36

The studies cited above provide a good base to study the effect of hydroxyurea on
RBC derived ATP release. It is well established that ATP, a known stimulus of
endothelial derived NO synthesis, is released in response to mechanical deformation of
the RBC membrane. Therefore, if hydroxyurea has an effect on RBC deformability, there
is a good possibility that it has an affect on RBC derived ATP release. None of the
studies in the literature have shown the effect of ATP release from RBCs in response to
hydroxyurea.

In line with expectations, as described in chapter 3, RBCs released higher
amounts of ATP under mechanical deformation in response to hydroxyurea treatment.
However, the amount of ATP release varied with hydroxyurea concentration, reaching a
maximum at 100 11M hydroxyurea concentration in a 7% hematoctrit of RBCs. These
studies suggested the possibility of changing deformability of RBCs in the presence of
hydroxyurea, as ATP release is an indirect way of measuring RBC membrane
deformability. However, the exact mechanism of hydroxyurea affect on RBC derived
ATP was not clear from these studies. The same trend for ATP release from RBCs was
observed for the NO treated RBCs suggesting the ability of hydroxyurea to form NO.
Furthermore, there have been studies showing the effect of NO on RBC deformability.
However, the controversial findings of the effect of NO on deformability have taken

away the potential significance of this effect. Therefore, experiments were carried out to

see the effect of NO on RBC derived ATP release. Interestingly, the same trend as that

119

seen with hydroxyurea was observed for NO treated RBCs as well. This study led the
hypothesis that NO formed from hydroxyurea is responsible for the increased ATP
release from RBC in response to mechanical deformation. Considering the reactions of
NO formation by hydroxyurea, it is further hypothesized that hydroxyurea can induce
NOS activity of RBCs thereby affect the NO production in RBCs. Moreover, the
produced NO can improve the RBC membrane deformability thereby increase the release

of ATP from RBCs.

4.4 Experimental

4.4.1 Methods and materials

Generation of washed red blood cells

RBCs were prepared on the day of use. For obtaining rabbit RBCs, male New
Zealand White rabbits (2.0-2.5 kg) were anesthetized with ketamine (8.0 mg/kg) and
xylazine (1.0 mg/kg) followed by pentobarbital sodium (15 mg/kg iv). After tracheotomy,
the rabbits were mechanically ventilated (tidal volume 20 ml/kg, rate 20 breaths/min;
Harvard ventilator). A catheter was placed into a carotid artery, heparin (500 units, iv)
was administered, and after 10 min, animals were exsanguinated. Blood was collected
into vials and the RBCs separated from other formed elements and plasma by
centrifugation at 500 x g at 4 °C for 10 min. The supernatant and buffy coats were
removed by aspiration. Packed RBCs were resuspended and washed three times in PSS

[PSS; in mM; 4.7 KCl, 2.0 CaClz, 1.2 MgSO4, 140.5 NaCl, 21.0

120

tris(hydroxymethyl)aminomethane, and 11.1 dextrose with 5% bovine serum albumin,

pH adjusted to 7.4].

4.4.1.1 Chemiluminescence detection of ATP

Preparation of A T P standards

All the reagents were purchased from Sigma chemical company, unless otherwise
noted. A 100 11M stock solution of ATP was prepared by adding 0.0551 g of ATP to 1000
ml of distilled and deionized water (DDW). The standard solutions (0, 25, 50, 100, 150
nM) were made by adding 0, 67.5, 125.0, 187.5, 250.0, and 375.0 pl of 100 11M ATP
stock solution to PSS. All the standards were made in 25 ml volumetric flasks and made
the day of use. PSS was made by first combining 25 ml of Tris buffer and 25 ml of
Ringer’s buffer and then adding 0.50 g of D-Glucose and 2.50 g of bovine albumin
fraction V (fatty acid free). The entire solution was then diluted to 500 ml by adding
DDW and adjusting the pH to 735-745. The PSS solution was filtered three times using
0.45 urn filter (Corning, Fisher Scientific) before use. All the standards were made on the

day use to calibrate the chemiluminescence signal on the PMT.

Preparation of luciferin-luciferase reaction mixture

Firefly extract (contains luciferin and luciferase) was dissolved in 5.0 ml of DDW. Next,
0.002 g of synthetic luciferin was added to the firefly mixture to enhance the sensitivity.
The chemiluminescence intensity generated by the ATP/luciferin-luciferase reaction was

measured.

121

Chemiluminescence measurement of deformation induced RBC derived A T P release

All RBC solutions were diluted to a 7% hematoctrit in PSS. To measure the RBC
derived ATP release, the luciferin- luciferase reaction mixture was placed in a 500 111
syringe (Hamilton, Fischer Scientific). The RBC solution was placed in another 500 pl
syringe (Hamilton, Fischer Scientific) and both syringes were loaded on to a syringe
pump (Harvard Apparatus, Boston, MA) and connected to two sections of silica
microbore tubing having an internal diameter of 50 um and an outer diameter of 362 um
(Polymicro Technologies, Phoenix, AZ). The microbore tubes were then connected to a
T-junction (Upchurch Scientific, Oak Harbor, WA) having a volume of 256 nl. The
combined contents were passed through a 6 cm microbore tubing having an internal
diameter of 75 pm. The resultant chemiluminescence produced by the RBC derived ATP
and the luciferin-luciferase reaction was detected by a PMT housed in a light excluding
box (shown in chapter 3). The polyimide coating was removed from the tubing and

placed over the PMT for measurement.

Non flow chemiluminescence measurements of RBC derived ATP release

The RBC solutions were diluted to 7% hematocrit and incubated in 50 11M
hydroxyurea for 20 minutes in normal PSS and calcium free PSS. The control 7% RBCs
were incubated in normal PSS and calcium free PSS. Next, 100 pl of incubated RBC
samples were mixed with 100 111 of luciferin-luciferase, followed by measurement of the
mix chemiluminescence intensity after 15 seconds by the PMT housed in the light

excluding box.

122

Other reagents

L-NAME: A 1 mM stock solution of L-NAME was prepared by dissolving 0.003 g of L-
NAME in 10 ml of phosphate buffered saline (pH 7.4)

Hydroxyurea: For studies involving hydroxyurea, a 4 mM hydroxyurea solution was
prepared by dissolving 0.004 g of hydroxyurea (Sigma Aldrich, St. Louis, MO) in 25.0
ml of PSS. The range of concentrations (0 — 200 nM) were made by diluting appropriate
volumes (0 — 500 pl) of the stock solution in a solution of 7% RBCs made in PSS. When
preparing the hydroxyurea treated RBCs, hydroxyurea was first added to PSS before
adding RBCs to create a homogenously treated RBC solution. RBC samples were
incubated for 20 minutes at room temperature before the measurement portion of the
analysis.

C-peptide: 0.25 mg of C-peptide was dissolved in 10 ml of DDW to make an 8.3 11M C-
peptide stock solution. Next, 250 111 of the stock solution is dissolved in 25 ml of DDW
and from that solution, 600 111 was taken to make the 10 nM working solution of C-
peptide. The working solution is dissolved in a zinc containing medium where the

concentration of both C- peptide and zinc are equimolar in solution.

4.4.1.2 Atomic absorbance spectroscopy (AAS) studies for calcium measurements in

RBCs

Preparation of calcium standards for atomic absorption spectroscopy

The Ca2+ standards (2.50, 25.0, 250.0, 2500.0 ug/l or ppb) were prepared by a

serial dilution of a 10,000 ug/l stock solution of standard CaCO3 in 25 ml volumetric

123

flasks. The CaCO3 stock solution was prepared by dissolving 0.004 g of CaCO3 in 1000

ml of 18.0 MQ DDW.

Preparation of RBC samples for atomic absorption spectroscopy

RBCs of a 7% hematocrit solution were incubated with 50 11M hydroxyurea for
20 minutes. Next, the RBC sample was centrifuged at 500 x g at 4 °C for 10 min to
remove the calcium containing supernatant. The packed RBCs were resuspended and
washed three times in calcium free PSS. Calcium free PSS was made by using calcium
free Ringers buffer, but the ionic strength was balanced by substituting an equivalent
amount of MgSO4 [PSS; in mM; 4.7 KCl, , 3.2 MgSO4, 140.5 NaCl, 21.0
tris(hydroxymethyl)aminomethane, and 11.1 dextrose with 5% bovine serum albumin,
pH adjusted to 7.4]. Finally the washed RBCs were lysed in DDW, and the membrane
ghosts were removed by centrifuging at 3000 x g at 4 °C for 10 min. The supernatant was

then taken for the AAS studies.

4.4.1.3 Electron paramagnetic resonance spectroscopy (EPR) for HbNO detection in

RBCs

Preparation of RBC samples for EPR
RBC solutions were diluted to a 7% hematocrit and incubated in 50 11M hydroxyurea for
20 minutes in normal PSS and calcium free PSS as needed. The incubated samples were

transferred to 3 mm quartz tubes and were frozen immediately in liquid nitrogen.

124

Preparation of N0 standards for EPR

The NO standards using Spermine NONOate were prepared as followed. The 38
11M spermine NONOate solution was prepared by diluting 10 111 of 38 mM stock
spermine NONOate solution in 10 ml of deoxygenated PBS buffer. Next, a series of
standard solutions (20, 40, 100, 200, 1000 11M) were prepared by using varying volumes
of 38 11M spermine NONOate solution in a total of 5000 ml deoxygenated PBS buffer.
The PBS buffer was deoxygenated in separate vials by the effervescence of nitrogen gas
for 4 minutes.

For some experiments, NO standards were prepared by dissolving gaseous NO in
deoxygenated PBS. A Schlink line vacuum system was employed in deoxygenating the
PBS 7.4 buffer. N0 gas was bubbled into the airtight flasks having deoxygenated PBS
buffer to make the stock solution of NO (1.9 mM). The series of dilutions were made by
diluting the appropriate volumes of the NO stock solution in deoxygenated PBS buffer in

air tight flasks.

EPR measurements and data processing

All the EPR measurements were carried out in a Bruker ESP-300E spectrometer
operating at 9.46 GHz using a Bruker ST4102 (TE102) microwave cavity. An Oxford
Instruments ESR-900 liquid helium cryostat together with a model ITC-502 temperature
controller was used to maintain the sample temperature at 4.2 K. The typical EPR settings
were as follows; power 5 mW, field modulation frequency 100 kHz, field modulation

amplitude 2.8 G, conversion time 327 ms, scan average 16. Each spectrum of either

125

spermine NONOate treated or hydroxyurea treated RBCs were obtained by subtracting

the EPR spectrum of untreated RBCs.

4.5 Results and discussion

The similar trend of ATP release in the presence of varying concentrations of
hydroxyurea and spermine NONOate led us to further investigate the NO producing
ability of hydroxyurea in RBCs. According to the hypotheses, NO is produced from
hydroxyurea, within RBCs, and the produced NO modulates the cellular deformability of
RBCs, thereby increasing the release of RBC derived ATP upon mechanical deformation.

EPR spectroscopy experiments have been used by several groups to demonstrate
in vivo and in vitro production of NO from hydroxyureau’24 In fact, EPR spectroscopy is
a reliable method of detecting and quantifying free radical species in biological systems.
The interaction of NO with hemoglobin results in the formation of the HbNO molecule
and is also paramagnetic. The detection of HbNO in RBCs has been used as an indicator
molecule for previous hydroxyurea studies.

Even though, it had been previously performed, a series of experiments were
carried out to investigate the ability of NO formation from hydroxyurea within the
concentration range used for the RBC derived ATP release measurements (25- 200 11M).
Figure 4.3 shows the chgaracteristic EPR spectrum for Fe2+- NO comeplex. The two
peaks represent the g values at 6 and 2, respectively. In our experiments we were focused
on the peak at g=2, where a large negative dip represents the heme nitric oxide complex.

First, an EPR experiment was carried out at 4 K to detect the HbNO signal in

126

RBC samples treated with hydroxyurea. NO reacts rapidly with deoxygenated
hemoglobin to give HbNO. However, in this case, the RBCs were not subjected to
deoxygenation in order to preserve the physiological conditions of the circulation. In the
circulation, the majority of RBCs (98%) is in the oxygenated state while a small
percentage (~l-2%) will be in the deoxygenated state. Considering the concentrations of
hydroxyurea (25-200 uM) employed in the ATP release studies, a 100 11M hydroxyurea
solution was first chosen to incubate with the RBCs and try to detect any HbNO
formation from hydroxyurea derived NO.

Figure 4.4 shows the EPR signal obtained for RBCs incubated with 100 11M
hydroxyurea for 20 min. The spectrum obtained for the hydroxyurea treated RBCs was
subtracted from the spectrum obtained from untreated RBCs to obtain the difference
spectrum, which is in fact showing the HbNO signal due to hydroxyurea. The subtraction
method was applied to eliminate the EPR signal for anycopper containing protein,
cerruloplasmin (g = 2.06 where g is the spectroscopic splitting factor) in RBCs as it
overlaps almost the same field where HbNO occurs. The difference spectrum was
compared with the difference spectrum obtained from RBCs incubated with a known
concentration of spermine NONOate (100 11M) (figure 4.5). The comparison of the two
subtracted spectra obtained from hydroxyurea and spermine NONOate is shown in figure
4.6 for comparison. The triplet hyperfine coupling and the g values at 2.01 are the
characteristic features of HbNO formation.”38

Subsequently, a series of NO treated RBCs were prepared (frozen immediately

after addition of varying concentrations of NO standard solution) to quantify the HbNO

signal in RBCs at 4.2 K. The standard calibration plot shown in figure 4.7 and 4.8

127

confirms the formation of HbNO in RBCs with varying NO concentrations. The EPR
experiment to detect the HbNO signal in RBCs treated with gaseous NO was successful
even though the triplet hyperfine coupling is not observed.

Unlike the NO standards, hydroxyurea did not produce a measurable HbNO
signal in the EPR spectrum. This may be either due to the lack of formation of NO from
hydroxyurea or the insufficient amount of NO formation in RBCs to be detected by EPR.
Unfortunately, current EPR techniques are not sensitive enough to measure the basal
levels of NO (< 200 nM) in biological tissues.39 If the 100 1.1M hydroxyurea results in
nanomolar levels of NO, this would not be the ideal technique to detect smaller NO
concentrations in RBCs. Moreover, the studies that have been carried out by other groups
have utilized millimolar concentrations of hydroxyurea to obtain detectable HbNO triplet
hyperfine structure. Therefore, at this point, these experiments neither prove nor disprove
our hypothesis as it is difficult to interpret the results with the noted experimental
limitations. The experiment was performed with a concentration of hydroxyurea that
resulted in the highest ATP release from the RBCs. Therefore, further experimentation

with was not continuied for the other hydroxyurea concentrations.

128

20000 —

1
15000 4

T
10000 ~

'1
5000 ~

Intensity (arb)

 

 

-5000 -

1
-10000 -

 

 

T

l
1000 1500

1 r

l ' I l ' l ' I
2000 2500 3000 3500 4000
Magnetic Field (Gauss)

Figure 4.3 An EPR spectrum showing the characteristic peaks for Fey-NO complex
at g = 6 and g = 2.

129

WWWWWW
2400 2600 2800 3000 3200 3400 3600 3800 4000

Magnetic field (Gauss)

Figure 4.4 Typical EPR spectra obtained from control RBCs (a) and for 100 11M
hydroxyurea treated RBCs (B). Spectrum C is the difference obtained by subtracting b
from a. The EPR settings were as follows; power 5 mW, frequency 9.46 GHz, field
modulation amplitude 2.8 G, conversion time 327 ms, temperature 4.2 K, scan average 4.

130

WWWWWW
2400 2600 2800 3000 3200 3400 3600 3800 4000

Magnetic field (Gauss)

Figure 4.5 Typical EPR Spectra obtained from control RBCs (a) and 100 11M
spermine NONOate treated RBCs (b). Spectrum (c) is the difference obtained by
subtracting (b) from (a). A peak at g=2.01 represents the HbNO signal.The EPR settings
were as follows; power 5 mW, modulation frequency 9.46 GHz, field modulation
amplitude 2.8 G, conversion time 327 ms, temperature 4.2 K, scan average 4.

131

4000
RBCs + hydroxyurea

yvllh4/"/~~/

*— RBCs + spennineNONOate

2000
0 A

-2000

F1

.4000
-6000 _

\

\

2.018__. ’9
‘

2.012——>

-8000

Intensity (arb)
9
9

-10000
-12000

2.002 __.

-14000

9:

-16000
-18000

-20000 i
3300 3320 3340 3360 3380 3400 3420 3440

Magnetic field (Gauss)

Figure 4.6 The difference spectra obtained for spermine NONOate treated RBCs and
hydroxyurea treated RBCs. The triplet hyperfine structure at g =2.0l is the characteristic
signal of HbNO in RBCs. The triplet hyperfine structure corresponding to the g values of
HbNO in NO treated RBCs is not significant in hydroxyurea treated RBCs. The EPR
settings were as follows; power 5 mW, modulation frequency 9.46 GHz, field modulation
amplitude 2.8 G, conversion time 327 ms, temperature 4.2 K, scan average 4.

132

myvm’w Wm ,2. MW—MNPV’Vx/‘v'vm/WWWMA A
W J

A.;-v. “WM JJWWWA ”Ax W*M B
V/vk-J'MIV/VV

[FTVIIIIII'leII'I'I1IUIIIUI7IITIITIiIIU|

3200 3250 3300 3350 3400 3450 3500 3550 3600
Magnetic field (Gauss)

Figure 4.7 Typical EPR spectra obtained for the calibration curve of NO treated
RBCs. A: untreated RBCs. Spectra B, C, and D were obtained from RBCs treated with
20, 40 and 100 11M spermine NONOate respectively. The EPR conditions were as

follows: power 5mW; frequency 9.46 GHz, sweep time 15 minutes, temperature 4.2 K,
scan average 4.

133

8000
7000

6000 //////.///
5000 //’

4000 //

Intensity (arb)

3000

2000

1000 /////://///

OlT—V UUUUUUU l IIIIIIIII l IIIIII W—WIVYTWTU'II' VVVVVVVV TT‘rV’VI‘
0 20 40 60 80 100

NO concentration (11M)

 

Figure 4.8 Calibration curve of HbNO signal obtained from the reaction of RBCs and
gaseous NO. The peak heights of the HbNO signals are plotted against the varying NO
concentrations of gaseous NO (20-100 11M).

134

The primary objective of this study was to investigate the mechanism of
hydroxyurea on erythrocyte derived ATP release. Therefore, an attempt was made to
investigate the effect of hydroxyurea on NOS activity in the RBCs. The RBCs are known
to have the NOS enzyme in order to help preserve the RBC deformability within its life
time of 120 days.40‘4l A 7% hematocrit of RBCs were first treated with 100 uM L-
NAME, a known inhibitor for the NOS enzyme, to inhibit the NO production in RBCs.
Next, another 7% hematoctrit of rabbit RBCs were incubated with 100 uM L-NAME for
30 minutes, followed by 50 11M hydroxyurea for 20 minutes. The incubated samples were
pumped through silica microbore tubing having an internal diameter of 50 pm. The ATP
release from the RBCs was measured using the flow based system described in chapter 3.

The reason for performing this experiment was to examine the effect of NO
inhibition on RBC derived ATP release. As shown in figure 4.9, the the ATP release was
lower in L-NAME treated RBCs, when compared to untreated RBCs, suggesting the
involvement of NO on erythrocyte derived ATP release. Interestingly, a decreased ATP
release from RBCs was observed for RBCs incubated with L-NAME, followed by
hydroxyurea, compared to hydroxyurea treated RBCs. Therefore, this study suggested
the involvement of hydroxyurea on NOS activity, and possibly RBC derived NO
synthesis. In order to investigate these observations in depth, the mechanism of NO
synthesis was taken into consideration. It is well known that RBCs carry eNOS or the
constitutive form of NOS. The calcium-calmodulin (Ca-CAM) complex formation is the

first step in the activation of eNOS pathway in cells.

135

 

0.8 {

Normalized ATP release
3

0.6 f

0.4 {

 

 

 

 

 

 

 

0.2 '

 

 

7% 7%" HU 1°/o+ L'NAM'TO/o+HU+LNAM$V°+LNAME+HU

Figure 4.9 Measurement of ATP release from RBCs in the presence of L-NAME and
hydroxyurea. RBCs incubated with L-NAME resulted in the lowest release of ATP from
RBCs (third bar) showing the importance of intra erythrocyte NO production on ATP
release. Hydroxyurea did not have any effect on ATP release in the presence of L-NAME
indicating an important function of hydroxyurea on NOS in RBCs. The values between
(*) and (**) are significantly different (P< 0.04). n = 4 rabbits and the error bars are
represented as SEM.

136

In order to investigate the NO synthesis in RBCs in detail, an experiment was performed
to examine whether there is an effect of the calcium content on RBC derived ATP
release. Figure 4.10 shows the ATP release from RBCs in response to hydroxyurea in
PSS and calcium free PSS. There is no net effect on RBC derived ATP release for
hydroxyurea treated RBCs; compared to normal RBCs in calcium free PSS. This
implicates the involvement of calcium in the mechanism for hydroxyurea activated RBC
derived ATP release. However, this study did not confirm or eliminate the possibility of
an overall impairment of biochemical mechanisms in the absence of calcium. In order to
determine the impact of calcium ions on hydroxyurea activity, a calcium free buffer was
used to investigate the cellular mechanisms. ATP release was measured in the presence of
C-peptide in calcium free PSS. C-peptide has been shown to increase the ATP release
from RBC via a glucose uptake pathway. As expected, C-peptide treated RBCs released
higher amounts of ATP, compared to normal RBCs, confirming that the observed
increase in ATP release from RBCs with hydroxyurea in calcium free buffer is not due to
an impairment of all the physiological processes in the absence of calcium (data not
shown).

Another experiment was designed to measure the intracellular calcium
concentrations in RBCs. Iinitially, the calcium concentration was measured in using
fluorescence spectroscopy with an intracellular calcium fluorophore (fluo-4 AM cell
permeate, Molecular Probes). However, these experiments did not produce sufficient
fluorescence and the differences of the fluorescence intensity between samples were not

significant. This may be due to the matrix effects of the RBCs or due to the low

137

 

0.6

- 7%

- :37%+50uMHU .
0.5-

 

it

 

 

,‘ .....

ATP release (11M)
9
0)
—-—l

0.2 -

,‘\ .....

0.1 -

 

 

 

 

 

 

 

 

 

 

 

0.0 I I -
PSS Calcium free PSS

Figure 4.10 Measurement of the ATP release from RBCs in the presence and absence
of calcium ions in the medium. Black bars represent the ATP release of untreated RBCs
and gray bars represents the ATP release from RBCs treated with 50 11M hydroxyurea.
Increased ATP release is observed for hydroxyurea treated RBCs in PSS while no
significant increase of ATP release from RBCs is observed for RBCs treated with
hydroxyurea in the calcium free PSS. The data represented as (*) and (**) are
significantly different in P< 0.01. n = 5 rabbits and the error bars are reported as SEM.

138

sensitivity of the instrument. Therefore, atomic absorption spectroscopy (AAS) was
employed as it is a standard method for calcium quantification with high sensitivity. The
RBCs were first incubated with 50 11M hydroxyurea for 20 minutes. Lysed RBC samples
were then prepared as described above and determined by AAS. The figure 4.1] shows
an increase of calcium uptake in the presence of hydroxyurea in comparison to the
untreated RBCs. Calcium concentrations for RBCs were obtained from the calibration
curve using different calcium concentrations. The values shown in figure 4.11 represent
the calcium content of a 10 % hematocrit of RBCs. Taking the hematoctrit and the
volume of the RBC (80 fl) into consideration, the RBC calcium concentration was
calculated. The concentrations were 67 and 105 nM for RBCs in calcium free PSS and
normal PSS, respectively.

This observation was further investigated using EPR spectroscopy as the initial
EPR studies were carried out in phosphate buffered saline (PBS pH 7.4) where the
calcium concentration was zero. RBCs were incubated with 50 uM hydroxyurea for 20
minutes in normal PSS and calcium free PSS. Interestingly, a higher HbNO signal was
observed for RBCs in the normal PSS, where calcium is present, compared to the RBCs
that were in calcium free buffer. The signal subtraction method was employed here to
eliminate the interference from the cerruloplasmin protein (figure 4.12, and 4.13). A
comparison of the difference spectra obtained for hydroxyurea treated RBCs in calcium
free PSS and normal PSS is shown in figure 4.14. The triplet structure obtained at g = 2.0
confirmed the presence of HbNO signal for calcium containing buffer, suggesting the
involvement of Ca2+ in the NO formation of hydroxyurea. The absence of calcium in the

medium could be the reason for not initially detecting the HbNO signal for RBCs treated

139

 

 

 

Calcium concentration (ppb)

 

 

 

 

 

 

 

7% 7% + 50 pM HU

Figure 4.11 Atomic absorption spectroscopic study of calcium quantification in RBCs.
The first bar (gray) represents the calcium concentration in the cytoplasm of hematocrit
of 10% RBCs. The second bar (black) represents the calcium concentration in the
cytoplasm of hematocrit of 10% RBCs incubated with 50 11M hydroxyurea solution for
20 minutes. The values are significantly different in P< 0.025. n = 7 rabbits and the error
bars are represented as SEM.

140

2400 2600 2800 3000 3200 3400 3600 3800 4000
Magnetic field (Gauss)

Figure 4.12 Typical EPR Spectra obtained from control RBCs (a) and for 50 uM
hydroxyurea treated RBCs in normal PSS (b). Spectrum (0) is the difference spectra
obtained by subtracting (b) from (a). The EPR settings were as follows; power 5 mW,
modulation frequency 9.46 GHz; field modulation amplitude 2.8 G, conversion time 327
ms, scan average 16.

141

WWWWWW
2400 2600 2800 3000 3200 3400 3600 3800 4000

Magnetic field (Gauss)

Figure 4.13 Typical EPR Spectra obtained from control RBCs (a) and for 50 11M
hydroxyurea treated RBCs in calcium free PSS (b). Spectrum c is the difference spectra
obtained by subtracting (b) from (a). The EPR settings were as follows; power 5 mW,
modulation frequency 9.46 GHz, field modulation amplitude 2.8 G, conversion time 327
ms, temperature 4.2 K, scan average 16

142

5000 -

 

1.:- 1.

“’5..-
:1;-
3

i

2

s.-

A
.Q
I—
m d
V d
E
(D
C
a)
dd
5 4
-5000-
.. (”N
to
4 03 b
. NN
P
F
O.
N

 

-1 0000 JWWWWWW
3000 31 00 3200 3300 3400 3500 3600 3700 3800

Magnetic field (Gauss)

Figure 4.14 The difference EPR spectra obtained from RBCs incubated with 50 11M
hydroxyurea in calcium free PSS (a) and in calcium containing PSS (b).The triplet
hyperfine structure with g values at 2.02 indicates the presence HbNO in spectrum (b).
However, the corresponding peaks of HbNO are not significant in spectrum (b). The EPR
settings were as follows; power 5 mW, modulation frequency 9.46 GHz, field modulation
amplitude 2.8 G, conversion time 327 ms, temperature 4.2 K, scan average 16

143

with hydroxyurea. However, the EPR data are in the preliminary level and require further
investigation.

The results obtained using AAS and EPR for calcium studies were initially
thought as a paradigm of the currently approved theory for the role of calcium in SCD.
Higher intracellular calcium concentrations (five to ten fold increase compared to normal
RBCs) are known to be a characteristic feature of sickle RBCs.42 Moreover, a higher
influx of calcium has been observed during the deoxygenation of sickle RBCs suggesting
a possible effect of high calcium levels on sickled RBC water loss or dehydration. In that
instance, a higher calcium uptake by hydroxyurea wouldn’t benefit nor improve the
rheological properties.

However, many studies have failed to demonstrate any measurable activity
increase of the K+ channel or calcium pumps in sickled RBCs compared to healthy
human RBCs. Therefore, it is difficult to find a correlation between calcium content and
the dehydrogenation of RBCs, or any other related pathological event associated with the
disease.43 In line with these observations, Lew et al proposed a possible explanation for
this behavior in 1985. According to their theory, sickle cell RBCs and to a lesser extent
normal RBCs carry calcium in intracellular vesicles.“'45 These endocytic vessels are
formed from the endocytosis of the RBC membrane and contain a calcium ATPase
pump.46 The compartmentalization of sickle cell calcium limits the available ionized
calcium levels in the cytoplasm, even though the calcium uptake was higher upon
deoxygenation.45 Most of the calcium in the intracellular vesicles is in the form of
precipitates of inorganic or organic phosphates. Furthermore, Murphy et al measured the

ionized calcium levels in oxygenated sickled RBCs, oxygenated normal RBCs, as well as

144

upon deoxygenation of sickled RBCs. This was an important study because the total
calcium content in the cells exists in three locations. The majority of calcium is found in
the cell membranes while lesser amounts are in the vesicle or in the ionized form in the
cytosol of the RBCs. There was no difference of ionized calcium concentrations between
oxygenated normal and sickle RBCs. Moreover, there was no significant increase in the
ionized calcium content in the cytoplasm upon deoxygenating sickled RBCs.47

The calcium containing intracellular vesicles are a characteristic feature of sickle
cells. Therefore, it is possible to suggest that these calcium vesicles are not involved in
the signaling pathways or other biochemical pathways in sickled RBCs. Intracellular
calcium is important in the NO synthesis in all the cell types containing the constitutive
form of NOS and RBCs are not an exception. Therefore, an influx of calcium can
increase the formation of calcium-CAM complex, thereby increasing the NO synthesis
within the cell.

The results obtained from these studies clearly demonstrate the influence of
hydroxyurea on NOS synthesis. Moreover, the AAS studies provide a novel mechanism
of hydroxyurea on erythrocytes. The ability of increasing the calcium content in RBCs is
intriguing and novel and provides a good correlation with NOS activity. Therefore, it is
possible to conclude that hydroxyurea increases the intracellular calcium concentrations
in RBCs, thereby increasing the synthesis of NO due to an increase in the calcium-CAM
complex. An earlier study has shown the ability of hydroxyurea to increase the
intracellular calcium concentration in endothelial cells by immobilizing the calcium pools
in the endoplasmic reticulum. However, the work here shows for the first time that an

increase in calcium influx occurs upon addition of hydroxyurea to RBCs. Furthermore,

I45

this study suggests a new theory on the formation of NO by hydroxyurea. In addition, this
work supports the findings of both increased and decreased deformability of RBCs upon
hydroxyurea and NO addition. When the hydroxyurea concentration is optimal, it
stimulates the NO synthesis by increasing the intracellular calcium concentration, which
in turn increases the cellular deformability. Actually, the reason of modulating the
deformability of RBCs with hydroxyurea is via NO production. NO has the ability to
interact with the membrane bound hemoglobin molecules and this interaction can affect
the membrane deformability. However, when NO production is high in response to high
doses of hydroxyurea on RBCs, reactive nitric oxide species (RNS) can be formed,
affecting the overall deformability of the cell membrane. RNS species, as well as NO,
oxidize membrane proteins, especially the spectrin molecules, thereby affecting the cell
membrane. Moreover, it is also known that NO itself acts to inhibit the activity of NOS
via a negative feedback inhibition pathway.

Even though the deformability was not measured directly in this study, the whole
explanation of deformability was supported by the results obtained from chapter 3. A
change in ATP release from RBCs with different concentrations of hydroxyurea and NO
suggested a change in deformability of RBCs. The work in this thesis explains the
possible mechanism of action of hydroxyurea on erythrocytes in regulating blood flow in
SCD. The NO, a known local vasodilator in the circulatory system, is being produced by
hydroxyurea helping to maintain sickled RBC deformability, which is otherwise lost.
This could lead in the uninterrupted blood flow in the capillaries and arterioles even in

deoxygenated conditions.

146

4.6 Conclusions

SCD is a genetic disease with a significant rheological impairment of the vascular
system. Hydroxyurea is a proven therapy for SCD and the mechanisms of the action of
the clinical improvements of the treatment have yet to be elucidated.

EPR and AAS studies along with the chemiluminescence data have shown the
effect of hydroxyurea on cytosolic calcium concentrations in RBCs. The higher calcium
influx within the cytoplasm would increase the NO production by NOS in RBCs as
calcium is an important component of the NO synthesis. Previous studies have shown the
effect of NO on RBCs deformability. In fact, RBCs release ATP in response to
mechanical deformation. Therefore, the observed ATP release from RBCs in response to
hydroxyurea could be the interaction of NO with the RBC membrane, thus resulting the
ATP release from RBCs. However, this study did not measure the RBC deformability
directly, but the fact that ATP release in response to mechanical deformation suggest that
the deformability is responsible for the modulation of the ATP release of RBCs. Overall,
the studies in this chapter 3 and 4 provide evidence for a novel mechanism of

hydroxyurea on erythrocytes in relation to the improvement of the blood flow.

147

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CHAPTER 5

Overall conclusions and future directions

5.1 Overall conclusions

The erythrocyte is a highly specialized but a very simplified cell. In fact, the lack
of a nucleus and the intracellular organelles are especially designed for efficient oxygen
and carbon dioxide transport through out the body by means of intracellular hemoglobin
molecules. In that respect, the function of red blood cells (RBCs) have been
oversimplified to oxygen transport, but these cells possess number of important metabolic
pathways for important physiological processes and for their survival. Glycolysis, and the
pentose phosphate pathway are two such pathways that exist in the RBCs for the
production of ATP and NADPH. In addition, RBCs maintain cellular deformability via
the strong antioxidant system present in the cells to allow passage through the small
capillaries without damaging the cell.

It is very well accepted that RBCs release ATP, a known stimulus for NO
synthesis, in response to mechanical deformation“;3 It has also been demonstrated that
RBCs are a required component of the vasodialation via its ability to stimulate NO.4‘S
Both these findings have become important in identifying a novel role of RBCs in
relation to regulation of the blood flow in small arteries and capillaries. Therefore,
identification of the metabolic pathways or factors that regulate the RBC derived ATP

release is necessary for better understanding this important and novel role of RBCs.

154

However, experimentation with RBC metabolic processes is challenging and
difficult due to the complex matrix found in RBCs. We have developed a new fluorescent
based method to quantify the major antioxidant in RBCs, glutathione (GSH), without a
separation of the cellular constituents.6 This method utilizes the previously known
fluorescence probe, MCB, in conjunction with the standard addition method. The
advantage of this method is its ability to quantify GSH in RBCs in the presence of
hemoglobin, a major interfering molecule of spectroscopic analysis of biological samples.
The value obtained for the bulk sample for the RBCs from n=6 different rabbits was
0.042 d: 0.002 mM. This number translates to a cellular concentration of 3.99 :l: 0.25
mM/RBC in the sample and 362 i 20 amol/cell. These values were within the acceptable
concentration range of the previously published values.

Moreover, this technique was successfully employed in the investigation of the
effect of antioxidant system in RBC derived ATP release. Our group was the first to
report quantitatively that the diabetic RBCs release less amount of deformation induced
ATP release in comparison to the ATP release of healthy non diabetic RBCs.7 It is well
established that diabetes RBCs are less deformable and also have a weakened antioxidant
system. But there was no single study that linked these two factors to the diabetes
complications. We believe that the decreased ATP release from diabetic RBCs directly
involve in the diminished NO production from endothelium thereby affect the regulation
of blood flow. This could be the reason for higher incidence of heart attacks, strokes, as
well as, higher probability of having pulmonary hypertension in diabetic patients. In that
respect, the development of a novel fluorescent based assay for glutathione measurements

in RBCs without separating the cellular material greatly contributed to establish the

155

relationship between the weakened antioxidant system found in diabetic patients and the
decreased ATP release in relation to the stiffening of RBCs membranes. The GSH
concentrations and the ATP release from RBCs subjected to similar oxidant stress were
monitored for over 45 minutes in 5 minute intervals. Interestingly, two graphs
demonstrated the similar trends suggesting the relationship between the oxidant stress
and the ATP release from RBCs. Moreover, this new techniques was a rapid and simple
method to investigate the effect of oxidant stressors on cellular antioxidant system within
the 45 minutes time period compared to the existing methods which could have taken
more than 18 hours for a single assay.

Therefore, the necessity of such a system is not only limited to accurate
glutathione measurement in RBCs, but also important in using in a large scale diagnostic
tool for disease diagnosis. Furthermore, this method is amenable to quantitative
determination of glutathione in a microfluidic chip which would be the ultimate useful in
metabolomic studies of RBCs. Also, this method could be used in quantifying glutathione
levels in a continuous flow system which could mimic physiological conditions of the
circulatory system.

The second part of the thesis is focused on investigating the effect of
hydroxyurea, a proven therapy for sickle cell disease, on erythrocytes. The hypothesis of
the modulation of ATP release from RBCs in response to hydroxyurea was based on the
literature of increased/decreased deformability of RBCs in the presence of hydroxyurea.
The micro flow technique with a chemilumincence detection was employed to determine
the effect of hydroxyurea on deformation induced ATP release from RBCs subjected to

varying concentrations of hydroxyurea for 20 minutes. The results obtained for this study

156

led further determination of the exact mechanism of action of hydroxyurea on
deformation induced ATP release. Investigating the NO forming ability of hydroxyurea
was the first step towards the postulation of the mechanism, and ATP release was
measured in the same way for RBCs incubated with varying concentrations of NO donor,
spermine NONOate. Interestingly, a similar trend was observed for both hydroxyurea and
NONOate treated RBCs, suggesting the involvement of NO on RBC derived ATP release
from hydroxyurea. Further experimentation provided evidence for the involvement of
hydroxyurea on the activity of NOS in RBCs. However, the mechanism of hydroxyurea
on NO production was not understood until the atomic absorbance spectroscopic studies
proved the increased uptake of calcium by RBCs in the presence of hydroxyurea. This
observation was firrther confirmed by EPR spectroscopy studies as NO peak was only
observed for hydroxyurea treated RBCs only in the presence of calcium

Therefore, we were successful in defining a novel role of hydroxyurea on
erythrocytes in sickle cell patients. The mechanism of the increased release of ATP from
RBCs is due to the increased uptake of calcium by RBCs in the presence of hydroxyurea
thereby, increasing the production of NO via the activation of RBC NOS enzyme. This
may be one of the mechanisms of the beneficiary effects observed in sickle cell patients
receiving hydroxyurea before any rise in HbF in RBCs. The increased ATP release from
RBCs could improve the local regulation of blood flow as ATP is a known stimulus for
the vasodilator, NO in vascular system. This would be a very important mechanism
explaining the reduced occurrence of vasoocclusive crisis, stroke, and heart attacks of

patient’s receiving hydroxyurea.8

157

In addition to defining a new mechanism of hydroxyurea on RBCs, this study
provided evidence for a new pathway of NO formation from hydroxyurea. The NO
formation via the activity of NOS in RBCs may be one of the possible reasons of
increased levels of NO metabolites in RBCs of patients receiving hydroxyurea.

However, the ATP release started decreasing when the hydroxyurea concentration
exceeded 100 11M. The reason of decreased amount of ATP release when the
hydroxyurea concentration is higher may be due to the overproduction of NO in the
RBCs. This could essentially develop increased oxidative stress in RBCs as NO can
rapidly reacts with oxygen species creating reactive nitrogen and oxygen species. This
could damage the RBC membrane and the deformability can be lost when the
hydroxyurea concentration is higher.

Overall, this thesis contributes towards the understanding of the importance of the
strong antioxidant system of RBCs in maintenance of the RBC membrane deformability
and RBC derived ATP releasef”7 Moreover, the work on this thesis defined a novel
possible mechanism of hydroxyurea on erythrocytes which could be helpful in regulating
the blood flow of sickle cell patients receiving hydroxyurea. Taken collectively, all these
findings were based on the detection and quantification of important metabolites or
elements such as ATP, GSH, HbNO, and calcitun found in RBCs which were once
known to be challenging and complex for analysis in the presence of hemoglobin.
Therefore, in addition to the elucidation of two important pathways of RBCs in relation
to disease pathogenesis and survival, this work greatly contributed towards the
development and utilization of simple, and rapid analytical method in quantification and

analyzing the biological samples in physiologically relevant conditions.

158

Taken collectively, this dissertation provides insight into the importance of
exogenous and endogenous metabolites/molecules or therapeutic agents on red blood cell
deformability and subsequent ATP release. Also, a development of a new technique of
glutathione measurement facilitates the simultaneous detection of GSH in microfluidic
devices which would be an ideal tool for disease diagnosis in future. Moreover,
understanding of the exact mechanism of action of hydroxyurea will be beneficial in SCD
treatment more efficiently and effectively and will guide to future aspects of SCD disease

management.

5.2 Future directions

The future remaining work of GSH studies is the analysis of GSH levels in RBCs
in the microfluidic chip. The multichannel microfluidic chip would be useful in multi
sample analysis of GSH and can be eventually employed commercially as a GSH
quantifying tool in disease diagnosis. There is not much to be done in that aspect other
than the optimization of the fluorescence detection under the fluorescence microscope in
the presence of RBCs.

However, for the studies involving hydroxyurea and RBCs, elucidating the
calcium channels responsible for the calcium uptake from RBCs in the presence of
hydroxyurea would be necessary as the calcium uptake pathway is still unknown. This
could be done by specifically inhibiting the existing calcium channels in RBCs and then
investigating the effect of ATP release from RBCs. These studies would give a better

understaning of the newly hypothsiszed mechanism of hydroxyurea on RBCs.

159

Moreover, much of the work has been carried out in rabbit RBCs. It is necessary
and important in repeating these studies in sickle cell RBCs to clearly demonstrate the
beneficiary effects of hydroxyurea on sickle cell RBCs of humans. It is assumed to obtain
the same results for rabbit RBCs and human RBCs due to the similarity of RBCs between
the two species. The experiments with the sickle cell RBCs would determine
quantitatively, the amounts of ATP release from RBCs in the presence of hydroxyurea.

Finally, an experiment that demonstrates the effect of hydroxyurea on NO
synthesis by endothelial cells would be advantageous in proving the proposed mechanism
of action of hydroxyurea on erythrocytes. This could be done in a microchip developed to
grow endothelial cells as well as to passage red blood cells through the microchannels.
An ideal chip design would be able to deform RBCs as they passage through the channels
and the released ATP due to the mechanical deformation should diffuse in the endothelial
cells grown on the chip, and stimulate the NO synthesis. The multi channels on the same
chip would allow the NO production from endothelial cells in untreated RBCs as well as
RBCs treated with different concentrations of hydroxyurea.

Also, a study that involves the demonstration of the interaction of RBCs and
endothelial cells in the presence of hydroxyurea could be done on a microchip based
model that mimics the circulation. This could be an important study as hydroxyurea is
known to reduce the adhesion of sickle RBCs to endothelial cells. Also, this is considered
as an additional mechanism of action of hydroxyurea on improved rheological properties

of the vascular system observed in sickle cell patients.

160

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