1

The Regulation of the Serotonin Transporter and Na+/K

+

ATPase Pump by Thrombin in Rat Brain Synaptosomes

Neur 40060

Marie Sheehan – 10308687

Supervisor: Dr. Jana Haase

Submitted in part fulfilment of the requirements for the B.Sc (Honours)

Degree in Neuroscience

UCD School of Biomedical and Biomolecular Science

2

Summary:

The serotonin transporter is a member of the SLC6 gene family, a group of plasma

membrane transporters. The serotonin transporter is responsible for the reuptake of the

transmitter serotonin into neurons, after its release from presynaptic terminals. The

serotonin transporter functions through the active transport coupled to Na+ /Cl

- and K

+

ion gradients across the plasma membrane. This uptake helps terminate signal

transmission and is the first step in neurotransmitter recycling from the synaptic cleft.

Studies have shown that thrombin reduces the level of SERT activity in platelets and

brain tissue, most likely through interactions with PAR receptors. This study was aimed

at investigating the effect of thrombin on the uptake activity of serotonin in rat brain

synaptosomes, and whether the inhibitory effect of thrombin is linked to the Na+/K

+

ATPase pump. To do this, synaptosomes were prepared from rat brains. Synaptosomal

uptake of [3H] - 5HT was measured by 5-HT uptake assays using freshly prepared

synaptosomes. From these tests, it was found that thrombin had an inhibitory effect on

the 5-HT uptake in purified synaptosomes. Lipid rafts were isolated through the

centrifugation of crude synaptosomes through a discontinuous sucrose gradient.

Following lipid raft fractionation and isolation, these samples were then analysed by

Western blot techniques, using an array of antibody probes. Further studies were carried

out on both crude and purified synaptosomes, namely the investigation of Na+/K

+

ATPase pump activity, which was measured through a colorimetric ATPase assay. The

activity of the Na+/K

+ ATPase pump was shown to be reduced also in both crude and

purified synaptosomes in the presence of thrombin. This suggests that thrombin may

affect SERT activity by inhibiting the Na+/K

+ ATPase pump and hence diminishing the

Na+ gradient required for SERT-mediated 5-HT transport.

3

Table of Contents

Summary

Introduction

1.1: The serotonin transporter 1

1.2: Na+/K

+ ATPase pump 3

1.3: Thrombin 4

1.4: Project aims 4

Materials and Methods

2.1: Materials 6

2.2: Methods 8

2.2.1: Synaptosomal preparations and treatments 8

2.2.2: Synaptosomal 5-HT uptake assay 9

2.2.3: Lipid raft fractionation 9

2.2.4: Western blot 9

2.2.5: Colorimetric ATPase activity assay 10

Results

3.1: Synaptosomal 5-HT uptake assay 12

3.2: Western blot 14

3.3: Colorimetric ATPase activity assay 16

Discussion

4.1: Effect of thrombin on 5-HT uptake 24

4.2: Thrombin and lipid raft fractionation 24

4.3: Thrombin effect on Na+/K

+ ATPase pump 25

Acknowledgements 26

References

Abbreviations

Appendices

4

Figures and Tables

Figure 1: 5-HT uptake plate layout

Figure 2: 5-HT uptake in crude synaptosomes

Figure 3: 5-HT uptake in purified synaptosomes

Figure 4: Western blot examining thrombin treated lipid raft fractionation, using SERT,

flotillin, α3 and β1 ATPase antibodies

Figure 5: Western blot examining the effect of MβCD treatment on lipid raft fractions

Figure 6A: ATPase assay examining buffer absorbance

Figure 6B: ATPase assay examining fresh versus frozen ATP hydrolysis

Figure 7: 96 well plate assay using ATPase buffer and 34% sodium citrate

Figure 8: Crude protein concentration range in 96 well plate

Figure 9: Purified protein concentration in 96 well plate

Figure 10A: Crude versus purified synaptosomes in 96 well plates

Figure 10B: Comparison between crude and purified synaptosomes oubain sensitive

ATPase activity

Figure 11A: Effect of thrombin treatment on Na+/K

+ ATPase activity in crude

synaptosomes

Figure 11B: Examination of the effect of MβCD on crude synaptosomes oubain

sensitive ATPase activity in ATPase buffer, carried out in 96 well assay format

Figure 12: Examining the effect of thrombin treatment on Na+/K

+ ATPase activity in

purified synaptosomes

Table 1: Components of polyacrylamide gel for western blot

Table 2: Western blot antibody type and concentration

Table 3: ATPase activity assay vial layout and composition

Table 4: Pi standard curve vial composition and concentrations

5

Introduction:

1.1: The serotonin transporter

The seotonin transporter is a key psycho-stimulant and antidepressant target in the brain,

and is responsible for the re-uptake of the neurotransmitter serotonin in neurons as well

as in a number of peripheral cells. The serotonin transporter is a monoamine plasma

membrane transmitter belonging to the SLC6 gene family, similar to dopamine and

noradrenalin transporters.1 SERT functions by trafficking serotonin into the nerve cell

with Na+ and Cl

-, and transporting K

+ out of the nerve in the same reaction. It represents

the first step in transmitter recycling, as it is responsible for the reuptake of exocytosed

transmitters in the synaptic cleft. 5-HT is stored in serotonergic neurons in the brain, but

it is also stored in blood platelets in the periphery. It is believed to play a vital role in

platelet activation, aggregation and shape change during blood coagulation.2 To explain

the serotonin transport mechanism, an understanding of the alternative access model is

required.3 This model involves the binding of a substrate on both the cytoplasmic and

extracellular side of the plasma membrane. This model allows the difference in a

substrate’s transmembrane concentration to be the driving force behind the exchange. In

5-HT transport, a conformational change can only occur once a 1:1:1 stoichiometry

binding of Na+, Cl

-, and 5-HT occurs. Once this binding has occurred, an alteration in the

transporter conformation results in the translocation of these molecules from the

extracellular space to the cytoplasm. These substrates then dissociate to the cytoplasm.

Then, the transporter binds cytoplasmic K+ ions, and reverts back to its original

conformation, releasing the K+ into the extracellular space.

1 The transporter is composed

of 12 transmembrane domains, connected by hydrophilic loops. In recent years, SERT

has been exploited as a therapeutic drug target in the treatment of neurological disorders,

predominantly depression. SSRIs, such as citalopram, paroxetine and flouxetine, operate

6

by blocking SERT, thus increasing the transmitter’s time spent in the synaptic cleft. It

has been found that tricyclic antidepressants, such as imipramine, block the reuptake of

serotonin in synapses, prolonging the time it spends in the synapses, and therefore

prolonging receptor activation. This SERT inhibition is a competitive mechanism.4 SERT

levels have been implicated in numerous major conditions, such as alcoholism, major

depression and impulsive aggressive behaviour.5 SERT has been found to be associated

with cholesterol rich lipid rafts domains, namely in detergent-resistant fractions with a

high concentration of lipid raft markers. When cholesterol levels are depleted, it was

found that SERT activity levels decrease.6 It has been proposed that SERT's association

with lipid rafts is a regulation mechanism for the activity of the transporter and serotonin

transmission in the CNS. The stabilization of the SERT structure requires membrane

cholesterol.7 SERT expression has been shown to be regulated through a number of

mechanisms, namely PKC activation, intracellular Ca+ level elevation, and by phorbol

12-myristate 13-acetate treatment. All of these mechanisms result in the down regulation

of SERT.7 SERT function has also been shown to be modulated through protein-protein

interactions, a primary one being the SNARE protein syntax 1A. It exerts it’s function

through the regulation of the number of SERT molecules expressed on the plasma

membrane.8 One particular interacting protein identified in Dr. Jana Haase's lab is the β1

subunit of the ATPase pump, suggesting a direct interaction between SERT and ATPase.

This led to the hypothesis that SERT and ATPase may directly interact and that the close

physical interaction may be required for efficient coupling of SERT with the Na+

gradient generated by the Na+/ K

+ ATPase pump. This close physical interaction may be

due to the presence of both SERT and ATPase in lipid microdomains. 5-HT transport by

SERT is crucially dependent upon the Na+ gradient, with inhibition of the Na

+/K

+

7

ATPase pump causing a reduction in SERT activity. This is due to the phosphorylation of

ATP being dependent upon sodium.9

1.2: Na+/K

+ ATPase pump

Na+/ K

+ ATPase pump is an important cellular component of the plasma membrane. It

functions to transport Na+ and K

+ ions across the plasma membrane using the hydrolysis

of ATP as the driving force.9 This process typically involves the translocation of 3 Na

+

ions out of the cell, with 2 K+ ions moving into the cell. This transfer generates both an

electrical and chemical gradient across the membrane. The pump is responsible for the

maintenance of the cell’s osmotic balance, the resting membrane potential, as well as the

excitability of muscle and nerve cells.10

The Na+/ K

+ ATPase pump is unique in that it is

specifically inhibited by cardiac glycosides, such as oubain. These inhibitors function by

binding to the enzyme’s extracellular side, thus inhibiting it. The pump is composed of 2

subunits, the α and β subunits respectively.11

Isoforms of both exist, with emphasis being

placed on the α3 and β1 subunits in this study. The α subunit typically has a mass of

around 113 kDa, while a mass of between 35 and 55 kDa is associated with the β subunit.

The α3 isoform is found solely in neural and cardiac tissue, with the β1 isoform being

ubiquitously expressed. It is the α subunit that is believed to contain the ATP binding and

phosphorylation sites, as well as being the location for the amino acids that are known to

be crucial for the binding of cardiac glycosides. The α cation selective gate is believed to

be due to the α1 subunit’s lysine rich NH2 terminus.12

The β subunit thus far appears to

play a role localising the ATPase to the plasma membrane, as well as ensuring normal

enzymatic activity.9

8

1.3: Thrombin

Thrombin is a member of the serine protease family and may play a role in the

development and pathology of the nervous system. Although found extensively in the

periphery, where it has a major role in the coagulation cascade, recent studies are

focusing more on thrombin’s activities in the CNS.13

It has been widely implicated in

signalling cascades during ischemia and stroke, but the mechanisms of action in these

situations are still yet to be fully elucidated. The serine protease family is a diverse group

of enzymes that rely on a serine residue for nucleophilic catalysis.14

Thrombin is a

protein composed of 2 chains, A and B, which are linked via a disulfide bond. The A

chain is composed of 36 amino acid residues, while the B chain has 259 residues, within

which lies the serine protease domain.14

Studies are beginning to highlight the presence

of thrombin not only on platelets but also within the nervous system. It has been shown

previously that thrombin has the ability to enhance neuronal activity.15

This spontaneous

activity is believed to be linked to the onset of seizures through the activation of PAR1.16

It has been shown that under normal physiological conditions several cell types express

receptors to thrombin. Expression of thrombin receptors has been shown in both cultured

neuronal cells, as well as in rat brains. Receptor localization in the brain and the spinal

cord has also been achieved, with specific areas on neurons and glia highlighted. This

receptor is being linked to an increase in intracellular calcium levels due to its influence

on the hydrolysis of inositol phospholipid. Proteases such as thrombin are believed to

regulate cells through the cleaving of PARs, with thrombin having a specific effect on

PAR1.17

PAR1 has been shown to have a modulatory role in the brain, with an effect on

the plasticity and transmission of synapses. PAR1 belongs to a group of G-protein

coupled receptors, and activation of this receptor required the cleavage of a peptide

bound on the extracellular N-terminus.18

9

1.4: Project aims

The aim of this project was to investigate the role of thrombin in the regulation of the

serotonin transporter and Na+/K

+ ATPase pump in rat brain synaptosomes. Synaptosomes

are isolated nerve terminals, formed through the homogenisation and centrifugation of

neural tissues. It has been recently shown that activation of PAR receptors by thrombin

results in the inhibition of SERT activity in platelets, but also in cultured mammalian

cells and rat brain synaptosomes.19

The aim of this project was to study the mechanism of

action of thrombin in rat brain synaptosomes in greater detail. A particular focus was on

the potential effects on the activity of the Na+/K

+ ATPase pump, which is responsible for

the maintenance of ion gradients across the neuronal membrane, and is also critical in

providing the driving force for the Na+-dependent transport of serotonin.

10

Materials and Methods:

2.1: Materials

[3H]-5-HT PerkinElmer

Acrylamide National Diagnostics

Adenosine triphosphate Sigma

Ammonium molybdate Sigma

APS Sigma

Ascorbic acid Sigma

BCA assay kit Pierce

Brij-58 Sigma

CaCl2 Sigma

DTT Sigma

ECL Pierce

EDTA Sigma

Escitalopram Sigma

Glucose Sigma

Glycine Fisher

HEPES Sigma

Horse serum Sigma

KCl Sigma

Malachite green Sigma

Methanol Sigma

MgCl2 Sigma

NaCl Sigma

Nitrocellulose membrane Sigma

11

Oubain Sigma

Polyvinyl alcohol Sigma

Potassium dihydrogen phosphate Sigma

SDS Sigma

Skim milk powder Sigma

Sodium citrate Sigma

Sucrose Sigma

TEMED Sigma

Thrombin Sigma

Trizma Base (Tris Base) Sigma

Tween 20 Sigma

2.1.1 Antibodies

Β-Actin (C4) Santa Cruz

Serotonin Transporter (C20) Santa Cruz

Donkey anti-goat Licor Biosciences

Goat anti-mouse Pierce

Flotillin BD Biosciences

ATPase α3 Pierce

ATPase β1 Millipore

12

2.2: Methods:

2.2.1: Synaptosome preparation and treatments

Crude synaptosome:

The entire protocol was carried out on ice, and all centrifugation steps were carried out at

4°C to slow protein degradation. On ice, the brain was removed and dissected; with the

cerebellum removed (this is due to the low SERT content in the cerebellum). The tissue

was weighed and then placed in 9X volume homogenisation buffer (O.32 M sucrose).

The brain was then quartered before homogenisation commenced. Homogenisation was

carried out by hand using a potter-elvehjem homogenizer. Initially a 10 mL potter was

used, but was swapped to a 5 mL potter. The homogenate was transferred to two 15 mL

centrifugation vials. The homogenate was then centrifuged at 1,300 X g for 10 min in a

centrifuge. Pellet P1 was discarded, and the supernatant S1 was transferred to a fresh

15 mL centrifugation vial. Supernatant was then centrifuged at 17,000 X g for 10min.

Supernatant S2 was discarded and pellet P2 was washed in 1 mL homogenisation buffer.

The resuspended P2 pellet was centrifuged at 17,000 X g for 10 min. The resulting

supernatant was removed and the pellet resuspended in either homogenisation buffer or

transport buffer (10 mM HEPES, 150 mM NaCl, 2 mM KCl, 1mM CaCl2, 10 mM

glucose, pH 7.4) to be used in further experiments.

Purified synaptosomes:

After washing the P2 pellet (above) and centrifugated, the pellet was resuspended in

homogenisation buffer. Next the homogenised pellet was layered on a discontinuous

sucrose gradient. This gradient was composed of 1.2 M, 1 M and 0.85 M sucrose, with

the homogenate layered on top. Two gradients can be made per rat brain. Beckman

13

centrifuge tubes (no: 355631 tubes) were used, with 0.32 M sucrose used to balance the

contents before centrifugation. Centrifugation was carried out at 82,500 X g for 2 hr at

4 °C, with acceleration and deceleration set to slow. After centrifugation, the top layers

were removed, with synaptosomes being recovered from between 1 M and 1.2 M layers.

Synaptosomes were washed with 10 mL of 0.32 M sucrose, and centrifuged at 17,000 X

g for 15 min. The resulting pellet was resuspended in transport buffer, with a BCA assay

being carried out to determine protein concentration. Volume was adjusted as necessary,

and synaptosomes were then treated or frozen and stored at -80 °C.

Treatments:

Thrombin:

Synaptosomes were prepared as per protocol, with protein concentration determined by

BCA assay. Synaptosomes were diluted to 1 mg/mL in transport buffer for lipid raft

fractionation or ATPase buffer (5 mM MgCl2, 80 mM NaCl, 20 mM KCl, 40 mM Tris-

HCl, pH 7.4) for ATPase assay, and were then divided into 0.2 mL aliquots. Aliquots

were preheated to 37 °C for 10 min. 2 µl of 100 U/mL (final concentration 1 U/µL)

thrombin was added and samples were incubated at 37 °C for 10 min exactly. These were

then snap frozen using liquid nitrogen, and samples were stored at -80 °C until needed.

Controls were treated in parallel, with no thrombin being added.

MβCD:

Synaptosomes were prepared as per protocol, with the protein concentration being

determined by BCA assay. Synaptosomes were diluted to 1 mg/mL in transport buffer or

ATPase buffer. 0.5 mL sample and 0.5 mL control were vortexed at 17,000 X g for 10

min. The supernatant was removed and pellets were resuspended in the appropriate

14

medium. Control was resuspended in the appropriate buffer, while the MβCD sample

was resuspended in 1 mL buffer containing 10 μg/mL MβCD. The samples were

incubated at 37 °C for 10 min, and then centrifuged for 10 min at 17,000 X g. Pellets

were resuspended in 0.5 mL of the suitable buffer.

2.2.2: Synaptosomal 5-HT uptake

For 5-HT uptake a 24 well plate was used, with plate layout as follows. 50 µg of

synaptosomes were added to each well to a total volume of 200 µl. Rows 2-6 contained

2.5 µL of thrombin of varying concentration, ranging from 0.05-1 U/mL. The top well of

each row (A1-6) contained 2.5 µl escitalopram. The plate was then pre-incubated in the

water bath for 10 min exactly. At time 0 min, 50 µL of the 5-HT solution (transport

buffer with glucose, 500 µM ascetic acid, 250 nM 5-HT) was added to each well in row 1

and to subsequent rows at 30 second intervals. At 6 min, 0.8 mL ice cold stop buffer

(transport buffer, no glucose, 1 µM escitalopram) was added to each well in row 1, and

subsequent rows at 30 second intervals. Plate contents were then aspirated onto a filter

mat and left dry in fume hood overnight. Filter mats were placed in sample bags before 4

mL scintillation fluid was added and samples were counted using the microbeta counter.

15

Figure 1: 5-HT uptake plate layout.

Wells contain synaptosomes, escitalopram or thrombin of varying concentration. The top

well of each row (A1-6) contained escitalopram, with thrombin added to wells in a

concentration gradient manner, ranging from none to 1.0 U/mL. [3H] -5HT was added to

each well at time 0, and subsequent wells at 30 sec intervals. After 6 min, stop buffer was

added; contents were aspirated to a filter mat which is left dry overnight, with uptake

measured the following day.

2.2.3: Lipid raft fractionation

Following treatment, synaptosomes in transport buffer were pelleted by centrifugation for

10 min at 12,000 X g. The supernatant was removed and pellet was resuspended in the

non-ionic detergent buffer Brij-58. Samples were then incubated on ice for 30 min. These

samples were then spun at 17000 X g for 10 min, at 4 °C to remove any non-lysed debris.

The supernatant was transferred to a new tube. 0.5 mL of the lysate was mixed with

equal volume 80% sucrose in TN (10 mM Tris-Cl, 10 mM NaCl). This mixture was then

16

transferred to SW55Ti ultracentrifuge tubes. Samples were overlaid with 1 mL of 30%

sucrose in TN, followed by 0.5 mL of 5% sucrose in TN, forming a discontinuous

sucrose gradient. Tubes were balanced as required with 5% sucrose in TN before being

centrifuged for 17 hr at 134,000 X g at 4 °C. 8 fractions of 312 µL were isolated from the

top of the gradient. 150 µL of each fraction was mixed with 6X sample buffer (0.375 M

Tris pH 6.8, 12% SDS, 60% glycerol, 0.6 M DTT, 0.06% bromophenol blue), and then

stored at -20 °C. Fractions were then diluted further to reduce sucrose content before

being analysed by Western blot.

2.2.4: Western blot

Samples were mixed with the appropriate volume of 6X sample buffer and incubated at

37 °C for 10-15 min. Samples, with a protein marker on either side, were loaded onto a

polyacrylamide gel (4% stacking gel, 10% resolving gel, table 1) and run at 80 mV for 2

hr, and 100 mV for one hr, and then 120 mV until the sample dye had cleared the

resolving gel. Using wet transfer, the protein was transferred to a nitrocellulose

membrane at 160 mA for 1 gel, or 320mA for 2 gels. This transfer ran for 90 min. The

membrane was then blocked for an hour in approximately 20 mL of 5% milk in TBST

(50 mM Tris, 150 mM NaCl, 0.05% Tween 20). After this, the membrane was rinsed

three times in TBST for 15 min and once in TBS (50 mM Tris, 150 mM NaCl), then

incubated for 1 hr with the primary antibody in horse serum + TBST solution. The

membrane was then washed 3 times in TBST for 15 min each and once in TBS for 15

min also. The membrane was then incubated for 1 hr with the secondary antibody in 3%

horse serum in TBST/HRP. Following this, the membrane was washed 3 times for 15

17

min in TBST and once in TBS. The membrane was incubated in ECL for 1 min before

being allowed to expose photographic film for suitable exposure times.

Table 1: Components of polyacrylamide gel for western blot

Components 4% Stacking Gel 10% Resolving Gel

H2O (mL) 4.2 5.6

0.5 M Tris (mL) 2.1 0

1.5 M Tris pH 8.8 (mL) 0 3.5

10% SDS (µL) 84 140

30% Acrylamid (µL) 1.8 4.6

10% APS (µL) 84 140

TEMED (µL) 14 14

Table 2: Western blot antibody type and concentration.

Primary Antibody Concentration Secondary Antibody Concentration

C-20 SERT 1:2000 Anti-goat 1:1000

Flotillin 1:1000 Anti-mouse 1:1000

ATPase α3 1:2000 Anti-mouse 1:2000

ATPase β1 1:1000 Anti-mouse 1:2000

2.2.5: Colorimetric ATPase activity assay

Initial ATPase assays were carried out as described by Chan et al. (1986).20

Synaptosomes were diluted to 50 µg/mL in 2 mL transport buffer (10 mM HEPES,

0.15 M NaCl, 1 mM KCl, 1 mM CaCl2, 1 mM MgCl2 in dH2O) or ATPase buffer (5.0

mM MgCl2, 80.0 mM NaCl, 20.0 mM KCl, 40.0 mM Tris-HCl in dH2O, pH 7.4). 200 µl

was placed in each Eppendorf vial. (There are 24 tubes, 8 samples done in triplicate).

These 8 vials were made up as per table 3.

18

Table 3: ATPase activity assay vial layout and composition

Sample Synaptosomes Buffer ATP Oubain

1 10 µg TB None None

2 10 µg TB 2 mM None

3 10 µg TB 2 mM 1 mM

4 None TB 2 mM None

5 10 µg ATPase None None

6 10 µg ATPase 2 mM None

7 10 µg ATPase 2 mM 1 mM

8 None ATPase 2 mM None

For both the vial and 96 well assay, Pi mixed reagent was made up containing 1 part

ammonium molybdate dissolved in 6N HCl, 1 part polyvinyl alcohol dissolved in boiling

H2O, 2 parts H2O and 2 parts malachite green dye dissolved in H2O. Pi mixed reagent

was allowed stand at room temperature for 30 min, or until a golden brown colour had

developed, before use.

Once vials were made up according to the table (with no ATP added) they were

incubated for 15 min at 37 °C. After this incubation period, 2 mM ATP was added and

the samples were incubated once more for 5 min exactly at 37 °C. 1 mL of Pi mixed

reagent was added to each vial and the samples were centrifuged at 17,000 X g for 5 min.

1 mL of each sample was transferred to fresh cuvettes and the absorbance was read at

630 nm.

19

96 well plate protocol:

Synaptosomes were diluted in ATPase buffer to a concentration of 250 µg/mL. 50 mM

stock of oubain was prepared by dissolving oubain in heated H20, around 60 °C, and

stored in 50 µL aliquots until needed, protected from sunlight. These aliquots were

preheated to 70 °C before use. Synaptosomes were incubated for 15 min at 37 °C. After

incubation, 10 µL of 2 mM ATP was to each well. This was then incubated for 5 min

exactly. After incubation, 200 µL of Pi reagent was added to each well, followed

immediately by 25 µL of 1.16 M sodium citrate solution. The absorbance was read at 630

nm right away.

Pi standard curve:

Using potassium dihydrogen phosphate (MW 136.09,) 100 mM stock was made. From

this a working stock of 0.1 mM was prepared.

Table 4: Pi standard curve vial composition and concentrations

Tube 0.1 mM working stock (µl) H2O (µl) Pi concentration (µM)

1 0 1000 0

2 10 990 1

3 20 980 2

4 30 970 3

5 40 960 4

6 50 950 5

7 100 900 10

8 150 850 15

20

9 200 800 20

10 300 700 30

11 400 600 40

12 500 500 50

250 µl of each of the above solutions was used. These were incubated for 10 min at

37 °C. 1 mL of mixed Pi reagent was added, and solutions were vortexed and read at

630 nm. For 96 well plate assay, 50 µl of each concentration was used and the protocol

was followed as normal. After incubation, 200 µl of reagent was added to each well,

followed by 25 µl 1.16 M sodium citrate. Absorbance was read immediately.

Results:

3.1: Synaptosomal 5-HT uptake assay

To examine the effect of thrombin on synaptosomal serotonin uptake, fresh

synaptosomes were prepared, and a series of uptake assays were carried out using a

concentration gradient of thrombin. These uptakes were carried out using both crude and

purified synaptosomes, with the controls of each taken to be 100% uptake. Thrombin is

believed to decrease the uptake of 5-HT in synaptosomes due to it’s activation of PAR

receptors. Thrombin’s effect on PAR receptors is known to be concentration dependent,

requiring a low concentration. Initial experiments focussed on the effect of thrombin on

5-HT uptake in crude synaptosomes. From these experiments it was found that thrombin

did not significantly decrease the 5-HT uptake in crude synaptosomes. No statistically

significant results were obtained over the thrombin concentration gradient. Results of the

5-HT uptake can be seen in figure 2.

21

Contr

ol

0.05 0.

10.

20.

51.

0

0

50

100

150

Thrombin Concentration U/mL

5H

T U

pta

ke %

of

Co

ntr

ol

Figure 2: 5-HT uptake in crude synaptosomes.

SERT activity in crude synaptosomes was measured in the presence of a concentration

gradient of thrombin. Crude synaptosomes were prepared and incubated in the presence

of 2.5 µL of varying thrombin concentrations for 10 min. The SERT activity was

expressed as percentage of control. A slight decrease was noted at 0.05 U/mL in crude

synaptosomes, however SERT activity was found to not be significantly reduced in the

presence of thrombin. Escitalopram was added to obtain SERT specific activity. N=4

This experiment was then carried out as per protocol using purified synaptosomes. A

reduction in 5-HT accumulation could be seen in the presence of higher concentrations of

thrombin, such as 0.5 and 1 U/mL. These results proved to be statistically significant, as

can be seen in figure 3.

22

Contr

ol

0.05 0.

10.

20.

51.

0

0

50

100

150**

**

Thrombin Concentration U/mL

5H

T U

pta

ke %

of

Co

ntr

ol

Figure 3: 5-HT uptake in purified synaptosomes.

SERT activity in purified synaptosomes was measured in the presence of a concentration

gradient of thrombin. Purified synaptosomes were prepared and incubated in the

presence of 2.5 µL of varying thrombin concentrations for 10 min. The SERT activity was

expressed as percentage of control. SERT activity was significantly reduced in purified

synaptosomes at higher thrombin concentrations. (n=2, 0.5 U/mL p=0.0070, 1.0 U/mL

p=0.0011). Values presented as mean = SEM **P˂0.01.

3.2: Western blot

To investigate the effect of thrombin on SERT, Western blots were carried out upon lipid

raft fractionations. Lipid raft fractionations were obtained due to the association of SERT

with lipid microdomains. In order for successful SERT activity to be achieved, the

transporter must be included in the microdomain, indicating a regulatory mechanism of

SERT through lipid rafts. Lipid rafts were obtained through the preparation of crude

synaptosomes, treated with thrombin and the lysates were prepared using the non-ionic

23

detergent Brij-58. Lysates were then layered on a discontinuous sucrose gradient and

centrifuged. Once the lipid raft fractions were collected, diluted and mixed with sample

buffer, they were loaded onto a Western blot gel. The gel was run according to the

protocol, with various antibody probes used, and the blots were then developed. The

samples treated with thrombin are presented in figure 4, showing little change between

control and thrombin samples, except for the β1 subunit, which appeared to be

diminished in lipid raft fractions upon treatment with thrombin.

Control Thrombin

A B C A B C

Figure 4: Western blot examining thrombin treated lipid raft fractionation, using

SERT, flotillin, α3 and β1 ATPase antibodies.

Crude synaptosomes were prepared, treated with thrombin and centrifuged to produce

lipid raft fractions. These fractions were combined and run on an agarose gel. Row A

24

contains the top 3 fractions, row B contains fractions 4-6, and row C of each column

contains fractions 7 and 8. Gel was run as per protocol. Bands appeared at expected

sizes, as indicated, with little difference between control and thrombin treated, save for a

shift in the β1 subunit band.

Samples were also treated with MβCD before under-going lipid raft fractionation. MβCD

is used to deplete the cholesterol from plasma membranes, disrupting lipid rafts and

therefore reducing SERT function. After lipid rafts were isolated, they were run on gels,

as outlined in the protocol. There was little change found in the gels between control and

MβCD treated. A noticeable difference is that β1 appears to be lost from lipid raft

fractions in MβCD treated samples. This can be seen in figure 5.

Control MβCD

A B C A B C

25

Figure 5: Western blot examining the effect of MβCD treatment on lipid raft

fractions.

Crude synaptosomes were prepared, treated with MβCD and centrifuged to produce lipid

raft fractions. These fractions were combined and run on an agarose gel. Row A contains

the top 3 fractions, row B contains fractions 4-6, and row C of each column contains

fractions 7 and 8. Most bands appeared at expected sizes, save for MβCD treated

samples at the β1 fraction. It appears as though a shift has occurred from the higher to

the lower fractions, with little β1 being found in fractions 1-3.

3.3: Colorimetric ATPase activity assay

Initially, the ATPase assay was carried using vials to determine the ATPase activity in

synaptosomes. However, using this methodology produced results with high ATP

hydrolysis in the buffer solutions, as can be seen in figure 7A. This high background

made it difficult to distinguish between the background hydrolysis of ATP, and the

specific hydrolysis of ATPase synaptosomes. High background can be caused by a

number of factors, such as free Pi contamination. To reduce the background hydrolysis,

the experiment protocol was modified in a number of ways. First, the volumes were

reduced from 1 mL of solution absorbance being read, down to 275 µl, which allowed the

experiment to be carried out in a 96 well plate. Next, a colour stabilizer was added, in

this case sodium citrate. A number of experiments were carried out, in which the

percentage of sodium citrate was altered, as well as the time before reading of absorbance

was also manipulated. Through these experiments it was found that 34% sodium citrate,

added immediately after reaction was stopped and absorbance read immediately, gave the

most consistent results. Another factor that was considered in order to reduce background

hydrolysis was whether the ATP used was fresh or had been frozen. Experiments were

26

carried out to determine if this was a factor in the high buffer absorbance of reaction

buffer containing ATP. As can be seen in figure 6B, there was little distinguishable

difference between fresh and frozen ATP, with high buffer absorbance present in control

samples. Fresh ATP however gave higher absorbance values in samples when measuring

ATPase activity in synaptosomes, and was used throughout the following experiments.

These modifications resulted in the reduction of ATP hydrolysis in the buffer solutions

containing no synaptosomes. Once the protocol had been optimized, the assay could then

be used to calculate oubain sensitive ATPase activity in both crude and purified

synaptosomes.

Buff

er

Buff

er +

ATP

0.0

0.2

0.4

0.6TB Buffer

ATPase Buffer

Ab

so

rban

ce (

630n

m)

Figure 6A: ATPase assay examining buffer absorbance.

ATPase assay carried out as per initial protocol, using vials. Transport buffer and

ATPase buffer were used to determine optimum buffer. Vials contained either buffer, or

buffer with 2 mM ATP. Vials were incubated for 5 min exactly at 37 °C. After this time,

1 mL of Pi mixed reagent was added to each vial to halt the reaction. High background

absorbance was seen due to the high ATP hydrolysis in both buffer solutions containing

27

ATP. ATPase buffer shown to be the optimum buffer and was therefore used in further

experiments.

To determine the cause of the high background ATP hydrolysis in buffer solutions, ATP

was made up fresh on day of experiment, and aliquots were prepared, frozen and

defrosted on day of experiment, to determine if it was a factor.

Syn

apto

som

es +

Buff

er

Syn

apto

som

es +

Buff

er +

ATP

Syn

apto

som

es +

Buff

er +

Oubai

n + A

TP

Buff

er +

ATP

0.0

0.5

1.0

1.5

2.0

Purified, fresh ATP

Purified, frozen ATP

Crude, fresh ATP

Crude, frozen ATP

Ab

so

rban

ce (

630n

m)

Figure 6B: ATPase assay examining fresh versus frozen ATP hydrolysis.

Vials were made up as per table 3. High background hydrolysis was observed regardless

of fresh or frozen ATP used in buffer with ATP solutions. Fresh and frozen ATP was

tested on both crude and purified samples. Fresh ATP was used after this experiment

due to better hydrolysis in both crude and purified samples. However it was determined

to not be a major factor in the high background absorbance.

28

In order to reduce the high background absorbance in buffer solutions, the protocol was

adjusted. ATPase buffer was the buffer chosen, with fresh ATP. 34% sodium citrate was

incorporated after the addition of the Pi mixed reagent, with absorbance read

immediately afterwards. Volumes were reduced from vials to a 96 well plate assay.

These adjustments let to the reduction in background absorbance due to reduced

hydrolysis of ATP in buffer solutions. Specific hydrolysis in synaptosome containing

solutions did not appear reduced.

Buff

er

Buff

er +

ATP

Cru

de Syn

ap +

ATP

0.0

0.5

1.0

1.5

Ab

so

rban

ce (

630n

m)

Figure 7: 96 well plate assay using ATPase buffer and 34% sodium citrate.

Protocol was optimized to reduce high ATP hydrolysis in buffer solutions. Protocol was

reduced to 96 well plate, with the inclusion of 34% sodium citrate, and absorbance read

immediately after addition. These alterations produced a reduction is in the background

ATP hydrolysis of the buffer sample.

29

Once the background absorbance had been reduced, the optimum protein concentration

had to be determined. This was done through carrying out the assay using an incremental

increase in protein concentration ranging from 2-10 µg. From this, the optimum protein

concentration could be determined.

0 5 100.0

0.2

0.4

0.6

0.8

Protein Concentration (ug)

Ab

so

rban

ce (

630n

m)

Figure 8: Crude protein concentration range in 96 well plate.

ATPase assay was carried out as per 96 well plate protocol, using a gradient of protein

concentrations of crude synaptosomes. Increased protein concentration resulted in

higher absorbance.

In order to determine the optimum protein concentration for the 96 well assay using

purified synaptosomes, the same protocol was followed as per the crude synaptosome

protein concentration assay. This was done through carrying out the assay using an

incremental increase in protein concentration ranging from 2-10 µg. Results obtained

indicated 10 µg purified synaptosomes to be the optimum protein concentration to use for

purified synaptosomes.

30

0 5 100.0

0.1

0.2

0.3

0.4

Protein Concentration (ug)

Ab

so

rban

ce (

630n

m)

Figure 9: Purified protein concentration in 96 well plate.

96 well assay carried out as per protocol using a concentration gradient of purified

synaptosomes. Increased protein concentration resulted in higher absorbance. 10 µg was

shown to give the highest absorbance in the linear range.

Next, 96 well plate assays were carried out to compare the absorbance generated with

crude and purified synaptosomes. Higher ATP hydrolysis was observed in crude

synaptosomes compared to purified synaptosomes. This may be due in particular to the

mitochondrial contamination in crude synaptosomes.

31

Bla

nk +

ATP

Cru

de ATP

Cru

de ATP

Oubai

n

Puri

fied A

TP

Puri

fied A

TP O

ubain

0.0

0.2

0.4

0.6

0.8

1.0

Ab

so

rban

ce (

630n

m)

Figure 10A: Crude versus purified synaptosomes in 96 well plates.

Fresh ATP and ATPase buffer were used to compare crude and purified synaptosome

absorbance. 96 well plate assay was carried out using10 µg crude and purified

synaptosomes. Higher absorbance values were obtained for crude synaptosomes

compared with purified. Background absorbance was much reduced using the optimized

protocol.

To further analyse the activity within the samples, the oubain sensitive ATPase activity

was examined. Higher activity was seen in crude samples compared to purified

synaptosomes. To isolate oubain sensitive ATPase activity, the assay was carried out

with some wells containing the cardiac glycoside oubain. Oubain is a known inhibitor of

the Na+/K

+ ATPase pump. Results from oubain containing wells were subtracted from

wells without (total ATPase), giving the final oubain sensitive activity, which

corresponds to the activity of the Na+/K

+ ATPase pump. Higher oubain sensitive activity

was observed in crude samples, illustrated in figure 11B.

32

Cru

de

Puri

fied

0

50

100

150

200

Ou

bain

Sen

sit

ive A

TP

ase A

cti

vit

y

(nm

ol

Pi/

mg

/min

)

Figure 10B: Comparison between crude and purified synaptosomes oubain sensitive

ATPase activity.

96 well plate assay carried out using crude and purified synaptosomes, with the cardiac

glycoside oubain present in some wells. Oubain inhibited the Na+/K

+ ATPase pump.

When results from oubain containing wells were subtracted from those without, oubain

sensitive ATPase activity could be seen. Crude synaptosomes exhibit higher oubain

sensitive activity compared to purified synaptosomes.

Following this, the effect of thrombin on the oubain sensitive ATPase activity was

examined. To examine the effect of thrombin, crude synaptosomes were treated with

thrombin. After treatment, a 96 well plate assay was carried out. Oubain sensitive activity

was calculated, and showed a slight decrease in activity in those synaptosomes treated

with thrombin, shown in figure 11A. However this difference was not found to be

substantial and additional experiments should be carried out to ascertain any effect.

33

Contr

ol Thro

mbin

Throm

bin

0

100

200

300

400O

ub

ain

Sen

sit

ive A

TP

ase A

cti

vit

y

(nm

ol

Pi/

mg

/min

)

Figure 11A: Effect of thrombin treatment on Na+/K

+ ATPase activity in crude

synaptosomes.

96 well plate carried out examining the effect of thrombin treatment on the activity of the

Na+/K

+ ATPase pump in crude synaptosomes. A slight reduction of oubain sensitive

ATPase activity can be seen in the presence of thrombin treated crude synaptosomes.

However, this reduction does not appear substantial.

34

Contr

ol MBCD

MBCD

0

200

400

600

800O

ub

ain

Sen

sit

ive A

TP

ase A

cti

vit

y

(nm

ol

Pi/

mg

/min

)

Figure 11B: Examination of the effect of MβCD on crude synaptosomes oubain

sensitive ATPase activity in ATPase buffer, carried out in 96 well assay format.

96 well plate assay was carried out using MβCD treated crude synaptosomes and control

crude synaptosomes. Oubain is used inhibited the Na+/K

+ ATPase pump. When results

from oubain containing wells were subtracted from those without, oubain sensitive

ATPase activity could be seen. A substantial reduction in oubain sensitive activity can be

seen in the presence of MβCD treated crude synaptosomes.

In this experiment MβCD was used as a positive control for lipid raft disruption. It has

been shown previously18

that thrombin inhibits SERT activity and lipid raft association.

It was hypothesized that this could be due to the disruption of the association between

SERT and ATPase, which may be functionally, and possibly physically, linked within

lipid rafts. Thus, MβCD was used to test whether lipid raft disruption would have an

effect on ATPase activity. This disruption affects the activity of the Na+/K

+ ATPase

pump, showing a substantial decrease in pump activity. This can be seen clearly in figure

35

11B, however, this experiment was carried out once and so would need to be replicated

to determine significance.

Purified synaptosomes were then treated with thrombin to investigate it’s effect on

Na+/K

+ ATPase activity in purified synaptosomes. From figure 12 it can be seen to have

a substantially reducing effect on the activity of the Na+/K

+ ATP activity.

Contr

ol

Throm

bin

0

50

100

150

200

Ou

bain

Sen

sit

ive A

TP

ase A

cti

vit

y

(nm

ol

Pi/

mg

/min

)

Figure 12: Examining the effect of thrombin treatment on Na+/K

+ ATPase activity

in purified synaptosomes.

96 well plate assay was carried out using control and thrombin treated purified

synaptosomes. Oubain sensitive ATPase activity was determined. Purified synaptosomes

Na+/K

+ ATPase activity was substantially reduced after thrombin treatment. Further

studies should be carried out to determine significance.

36

Discussion:

4.1: Effect of thrombin on 5-HT uptake

5-HT uptakes were carried out on both crude and purified synaptosomes, to examine the

effect of thrombin on 5-HT uptake. At varying concentrations, thrombin was shown to

have a reducing effect on the uptake of both crude and purified synaptosomes; however

in crude synaptosomes this result was not significant. Specific uptake levels were very

low compared to typical 5-HT uptakes. This may be due to the synaptosome preparation

method. In other uptakes the [3H] -5HT accumulation numbers are much higher than

what was achieved in the uptakes shown here (figures 2 and 3). This may be due to the

preparation method not resulting in highly active synaptosomes. In purified

synaptosomes, thrombin inhibited the 5-HT uptake significantly, as can be seen in figure

3. Overall the synaptosome preparation seems not to be optimal. An alteration in

synaptosome preparation may increase 5-HT uptake activity. However overall, even with

low 5-HT accumulation, an inhibitory effect of thrombin was still seen observed, in

particular in purified synaptosomes. This experiment would need to be replicated in order

to determine conclusively the significance of this reduction.

4.2: Thrombin and lipid raft fractionation

Crude synaptosomes were used in lipid raft fractionation. After the synaptosomes were

prepared and treated, with either thrombin or MβCD, lipid raft fractions were isolated.

Brij-58 was used as the detergent to lyse the synaptosomes before centrifugation. After

centrifugation, fractions were combined and ran on an agarose gel. Little difference was

observed between the control and treated samples. Thrombin appears to have no effect on

lipid raft formation; however in previous studies18

a change was seen. A similar result

was observed in samples treated with MβCD. A change is expected, as MβCD is a known

37

lipid raft disruptor, due to its removal of cholesterol from the plasma membrane.

Because of this, the results from the Western blot are inconclusive regarding the effect of

thrombin on lipid raft fractionation. It would be expected that MβCD, and to some

extend thrombin, would result in the disruption of lipid rafts. Only a slight change was

observed in the fractions analysed through Western blot. In both thrombin and MβCD a

shift in the β1 subunit observed. This indicates a disruption to the lipid raft association

with the β1 subunit. However, this experiment needs to be replicated in order to

determine the significance of this result and to quantify the shift in order to draw final

conclusions. This shift may be due to the disruption between the β1 subunit of the Na+/K

+

ATPase pump and lipid rafts in the synaptosomes. The β1 subunit has been previously

identified in Dr. Haase’s lab as a potential SERT- interacting protein, which led to the

hypothesis that SERT and the ATPase pump may directly interact. This may occur

through a close physical association, which may be required for the efficient coupling of

SERT with the Na+ gradient generated by the Na

+/K

+ ATPase pump. Previous work

19 in

platelets showed that thrombin causes a dissociation of SERT from lipid rafts, possibly

also disrupting the coupling to the ATPase pump.

4.3: Thrombin effect on Na+/K

+ ATPase pump

In order to analyse the activity of Na+/K

+ ATPase pump in synaptosomes, the initial

protocol had to be adjusted in order to reduce the background hydrolysis of ATP in

buffer solutions. The type of buffer and the freshness of ATP were shown to not be the

cause of the high absorbance. The reduction in volume, the addition of sodium citrate,

and the optimization of timing were essential in order to examine the accurate activities

of the Na+/K

+ ATPase pump, particularly in the presence of thrombin. The high

hydrolysis of ATP in buffer solutions was a major challenge faced initially, as this high

38

background masked the actual activity of synaptosome containing samples. The high

background made it impossible to accurately examine the oubain sensitive ATPase

activity in synaptosomes, also inhibiting the examination of the effect of thrombin on this

process. Once this challenge was overcome through the optimization of the protocol as

explained previously, thrombin was shown to reduce the activity of the Na+/K

+ ATPase

pump in the 96 well plate assays. This reduction was observed in both crude and purified

synaptosomes. To accurately examine the ATPase activities, the optimum protein

concentration of both crude and purified synaptosomes had to be determined. Once

again, the less than optimum crude preparation may have had a detrimental effect upon

these results, but it was uncovered that in purified synaptosomes, 10 µg of protein

produced the best results. In crude synaptosomes, a lower protein concentration, within

the linear range, would have been more suitable. Further experiments showed that

ATPase buffer and fresh ATP produced the best results in assays. One of the most

difficult aspects of this assay was the use of oubain. In order to function properly, oubain

must be heated to 70 °C before being pipetted, in order to ensure the oubain is in

solution. Considering the time constraints in the protocol, the handling challenges of

oubain may have had an effect on results. However, even with this challenge, thrombin

still exerted an effect on activity, yet the full extent of its effect may be masked by

oubain difficulties. Further experimentation should be carried out with a particular focus

on oubain handling which may produce more significant results. Further experiments

should also be carried out to determine fully the significance of the results illustrated in

figures 11A and 12, which show a reduction in oubain sensitive activity in the presence

of thrombin.

39

These results support the hypothesis that thrombin has an inhibitory effect upon the

serotonin transporter within rat brain synaptosomes, and decreases the activity of the

Na+/K

+ ATPase pump. Further research is required to establish the full extent of this

inhibitory effect, as well as determining through what mechanism this inhibition is

carried out. Further optimization of protocols is recommended in order to fully examine

the inhibitory effect of thrombin in synaptosomes, with a particular focus to be paid to

lipid raft fractions, due to the difficulty in obtaining conclusive results. However,

regardless of the challenges faced in regards to Western blot, conclusive results may also

be obtained through 5-HT uptake and ATPase activity assays regarding the effect of

thrombin in rat brain synaptosomes.

Word count:

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Abbreviations

APS Ammonium persulfate

DTT Dithiothreitol

SERT Serotonin transporter

PAR Protease activated receptor

42

DTT Dithiothreitol

ECL Enhanced chemiluminescence

EDTA Ethylenediaminetetraacetic acid

PBS Phosphate buffered saline

PKC Protein kinase C

SSRI Selective serotonin reuptake inhibitor

TBS Tris buffered saline

TBST Tris buffered saline with Tween 20

Appendices