the details of the materials and methods used in this...

The details of the materials and methods used in this study for the collection and

analysis of data has been described under the following heads:

A) materials used to collect the data

B) methodology used to acquire the data

C) Statistical analysis of data

3.1 MATERIALS USED TO COLLECT THE DATA

Animal procurement and maintenance

Male Wistar rats weighing 250-280 gms, obtained from the central animal

house facility of Jawaharlal Nehru University were used in this study. The animals

were kept in semitransparent polypropylene cages and were maintained under 12:12

dark: light cycle. Room temperature was maintained at 25-28 o C. Food and water were

supplied ad lib.

Equipments

Centrifuges and rotors used in this study were as follows:

a) A high speed ultracentrifuge capable of speeds upto 50,000 r.p.m.

b) A swing out rotor for the ultracentrifuge with the tube capacity of

1 Oml. Fixed angle rotors for ultracentrifuge 50 Ti and 80Ti.

c) A medium speed refrigerated centrifuge (Sorvall or REMI) capable of

speeds upto 15,000 r.p.m.

d) Fixed angle rotors for the medium speed centrifuge with the tube

capacity of30ml (SS-34) and 12ml (SM-24).

e) A bench top centrifuge (microcentrifuge) capable of speeds upto 6_000

r.p.m.

62

Homogenizer

A large homogenizer of the Potter- Elvehjam type having a glass mortar of

capacity about 30ml and a teflon pestle was used to homogenize rat brain tissue.

The pestle was motor driven with continous variable speed and high torque.

Spectrophotometer-UV2000 Hitachi or UV160A Shimadzu.

Spectrofluorimeter-RF 540 Shimadzu.

Chemicals

The list of chemicals, solvents and biochemicals used along with the name

of the supplier are as follows:

NAME OF THE CHEMICAL

Ammonium Molybdate

Ammonium Sulphate

ANSA

Arterenol (Norepinephrine

bitartarate)

ATP (Sodium salt)

Bovine Serum Albumin

Chlorotetracycline

Copper Sulphate

EDTA

EGTA

·Polin's Reagent

Imidazole

63

NAME OF THE SUPPLIER

CDH, India

SRL, India

CDH, India

Sigma.:Aldrich, USA

SRL, India

Sigma-Aldrich, USA

Sigma-Aldrich, USA

Qualigens, India

BDH, India

Sigma-Aldrich, USA

SRL, India

SRL, India

Lactic Acid Dehyrogenase

Magnesium Chloride

NADH

Ouabain

Phoshoenol pyruvate

Potassium Chloride

Potassium dihydrogen phosphate

Prazosin- HCl

Propranolol

Pyruvate kinase

Sodium Bisulphite

Sodium Carbonate

Sodium Chloride

Sodium Hydroxide

Sodium Potassium Tartarate

Sodium Sulphite

Sucrose

Thiobarbituric Acid

Trichloroacetic acid

Tris Buffer

64

Boehringer Mannheim,Germany

Qualigens, India

Boehringer Mannheim, Germany

EMerck, Germany


Qualigens, India

BDH, India

Sigma-Aldrich, USA

Sigma-Aldrich, USA


CDH, India

Qualigens, India

Qualigens, India

Qualigens, India

CDH, India

CDH, India

SRL, India

Sigma-Aldrich, USA

Qualigens, India

SRL, India

3.2 METHODOLOGY

REM Sleep Deprivation

In this study, a simple, non-surgical, non-invasive and relatively inexpensive

but effective method for selective REM sleep deprivation was used. This method is

known as water-tank or flower-pot or platform or pedestal technique and can be used

for short ~nd long term REM sleep deprivation. It also allows several animals to be

sleep deprived simultaneously. This method was first described by Jouvet et al.

( 1964) and has been now most widely used method for REM sleep deprivation (Hicks

et al., 1977; Oniani, 1988). This technique is well established in this laboratory.

In this procedure, the animals were maintained on small platforms surrounded

by water. On this artificial island, the animals could undergo SWS, however,

occurrence of REM sleep was restricted. As the animal entered into REM sleep, the

muscle tone was lost causing them to fall into the water and wake up. However,

within a short time the animal learns to wake up at the onset of REM sleep and avoids

falling into the water. This procedure allows the selective deprivation of REM sleep.

The experimental rats were maintained on 6.5 em diameter platform. The size

of the platform was selected based upon weight of the animals. Food and water were

supplied ad lib throughout. The animals were REM sleep deprived for 4 days. This

is because earlier studies from this lab showed that the period of four days was

optimum time required to express the effect in the REM sleep deprivation induced

increase in Na-K ATPase activity.

Free moving control (FMC) rats were kept in the same room m semi

transparent cages where the animals were kept for REM sleep deprivation. Food and

water were supplied ad lib. To rule out the possibility of non-specific effects, a group

of animals was maintained on a large platform of 12.5 em diameter. These large

65

platform control (LPC) animals were maintained in identical condition to that of the

experimental rats except that the platform size was a little larger so that the rats could

have both SWS and REM sleep. The larger platform control allowed the animals to

experience the same environmental stress as that of the small platform ones.

Biochemical Estimations

Biochemical estimations were performed in whole brain, as well as in different

areas of the brain from control and experimental animals .

After cervical dislocation, control and experimental rats were sacrificed by

decapitation. The skull was cut open and the brain removed within 3 minutes in a

petridish containing ice-cold homogenizing buffer. The tissue was then washed thrice

in chilled buffer to remove blood. After washing, either the whole brain or different

regions viz. cerebrum, cerebellum and brainstem were dissected on ice.

The tissue was homogenized in 10 volumes of buffer in the Potter-Elvjham

homogenizer (8-1 0 strokes) using different homogenization buffers depending on type

of biochemical estimation. Glass-double distilled water was used for preparation of

buffers. Homogenizing of the brain tissue was done in Sucrose-Tris buffer which

contained 0.32 M sucrose, 12mM Tris, pH 7.4.

Microsome and Synaptosome

Neurons are notable for their highly elaborate shapes. They possess long and

often branching processes which intermingle extensively with each other and with

glia. Such cells do not survive intact after homogenization. The cell bodies are

sheared from their processes which break up into small fragments. These plasma

membranes of cell fragments may reseal to form osmotically active particles called

66

microsome and when such particles contain the synaptic region, they ar~ known as

synaptosome.

Preparation of microsomes

The protocol used for microsome preparation is shown as a flow-chart in Fig

4. In order to study the effect of calcium on Na-K ATPase activity, the microsome

was prepared in the absence and presence of divalent cation chelator- Ethylene

diamine tetraacetic acid-EDT A.

The rat brains were homogenized in 0.32M sucrose, 12mM Tris-HCl buffer at

pH 7.4 with or without lmM EDTA. The homogenate was centrifuged for 5 min at

6000 r.p.m. (3000xg). The supernatant was centrifuged for 20 min at 12,000 r.p.m

·(11,000xg).The.supernatant obtained was ultracentrifuged for 60 min at 40,000 r.p.m

(150,000xg). The pellet obtained was suspended in 5mM Tris or 5mM EDTA, pH 7.4

(depending on whether the homogenization procedure was carried out in the absence

or presence ofEDTA, respectively) stirred for 30 min and centrifuged at 12,000 r.p.m

(ll,OOOxg) for 5 min. The supernatant was brought to 30% ammonium sulphate

saturation at pH 7.4 with Tris buffer. It was then stirred for 30 min at 4 oc and

centrifuged for 30 min at 12,000 r.p.m. (11,000xg). The final pellet obtained was

resuspended in 5mM Tris or 5mM EDTA (depending on whether non-EDTA or

EDTA conditions wer~ maintained earlier, respectively) and used as microsome for

the estimation ofNa-K ATPase activity in the unlysed sample. The sample was left

overnight at 4oC in Tris or EDTA buffer (as the case may be) for lysis and used as

lysed microsomal preparation for the estimation ofNa-K ATPase activity.

67

PREPARATION OF MICROSOME

BRAIN HOMOGENATE in 0.32M sucrose, 12mM Tris ± lmM EDTA, pH 7.4

l I SUPERNATANT I

12K I 20'

I SUPERNATANT'

40K I 60'

SUPERNATANT

6KI 5'

30% Amm. sulphate cut 12K I 30'

1 I PELLE_T .......

I PELLET

suspend in 5mM Trisl EDT A buffer, pH 7.4 12K I 5'

"PERNATANTI PELLET

UNLYSED MICROSOME

Fig4

suspend in 5mM Tris I EDT A buffer, pH 7.4.

LYSED MICROSOME

PREPARATION OF SYNA


I SUPERNATANT

12K I 20'

!sUPERNATANT

Suspend in buffer

I UNLYSED

SYNAPTOSOME

6KI 5'

[SU"PE_RN_ ATANT

Fig 5

PELLET I

PELLET

suspend in homogenizing buffer, ± EDT A 25K I 120'

BAND

f

Suspend in water

13KI 30'

I PELLET I

LYSED SYNAPTOSOME

Preparation of synaptosome

As in the case of microsome preparation, the rat brain was homogenized in

buffer containing 0.32M sucrose, 12mM Tris at pH 7.4 in the absence and presence

of 1mM EDTA. The rat brain homogenate was centrifuged for 5 min at 6000 r.p.m.

(3000xg) and the supernatant was centrifuged for 20 min at 12,000 r.p.m (11,000xg).

The pellet obtained was suspended in 1ml of homogenizing buffer (Fig 5).

The sucrose gradients were prepared in thin-walled rigid polycarbonate tubes.

Discontinuous gradient was made by layering individual concentration of sucrose on

top of one another carefully. 4ml of 1.2M sucrose was overlayed with 4ml of0.8M

sucrose. The gradient was prepared a few hours before use and left at 4 oc to allow the

interfaces to smear. The pellet suspension in homogenizing buffer was layered on to

the sucrose gradient and ultracentrifuged in a swing-out rotor (SW 41 or TH641) at

25,000 r.p.m. (1 OO,OOOxg) for 2 hours. The band obtained at the interface of 1.2M and

0.8M sucrose was carefully retrieved and diluted with homogenizing buffer. An

aliquot ofthis was used immediately as unlysed synaptosome for the estimation of

Na-K ATPase activity. The unlysed synaptosomal preparation was recovered by

centrifugation at 10,000 r.p.m. (8000xg) for 20 min and the pellet was subjected to a

hypotonic shock by suspending it in distilled water and leaving it overnight for lysis

at 4 oc. The suspension was centrifuged at 13,000 r.p.m. (13000xg) for 30 min and

pellet resuspended in distilled water. This was used as lysed synaptosome or

synaptosomal plasma membrane (Fig 5).

Estimation of Protein concentration

The or;•:

The protein concentration was estimated by the method of Lowry et al. ( 1951 ).

68

Protein reacts with Polin's reagent (Solution of sodium tungstate and sodium

molybdate in phosphoric and hydrochloric acid) to give a coloured complex. The

colour so formed is due to the reaction of copper with protein in an alkaline medium.

Alkaline copper sulphate reacts with compounds containing two or more peptide

bonds to give a violet coloured complex. The depth of the colour is a measure of

number of peptides present in the protein. Colour is also formed due to reduction of

phosphomolybdate by tyrosine and tryptophan present in a protein.

Assay System for protein estimation

The reaction mixture contained a suitable aliquot of the membrane preparation

or the homogenate and d~.mble distilled water was added to make up the volume to

0.5ml. 5ml of freshly prepared Lowry's reagent (Lowry's reagent contained 48 ml of

2% sodium carbonate dissolved in O.lNNaOH +lml of0.5% copper sulphate+ lml

of 1% Na-K Tartarate solutions) was added and the mixture was vortexed and

incubated at room temperature for 10 minutes. 0.5ml ofFolin's reagent (diluted to IN

with distilled water) was added, vortexed immediately and incubated for 30 minutes

at room temperature. The colour developed was read at 700nm in a Shimadzu UV 160

or a Hitachi 2000 spectrophotometer. Bovine serum albumin (IOmg/lOml) was used

as a standard.

Estimation of Na-K ATPase activity

The Na-K ATPase activity was estimated following the method by Akagawa

and Tsukada ( 1979). The method was adapted for our lab condition as reported earlier

(Gulyani and Mallick, 1993; Gulyani, S., Ph.D. thesis, 1994). The reaction buffer

contained 1 OOmM NaCl, 20mM KCl, 5mM MgC12, 3mM ATP and 50mM Tris, pH

7.4. An aliquot (20-30 ~g protein) from the microsome or synaptosomal preparation

69

was incubated with the reaction buffer at 37oC for 15 min. ATP was used as the

substrate and the liberated inorganic phosphate was estimated by Fiske and Subbarow

method (1925). ADP is produced as one of the products of the reaction. Since ADP

is inhibitory to the reaction, the reaction time was kept limited and constant so that not

more than 10-15% of ATP is hydrolysed . After 15 minutes, the reaction was stopped

by addition of 1 ml of 10% ice-cold trichloroacetic acid (TCA). Ouabain (1mM) was

used as a specific blocker ofNa-K ATPase activity. The ouabain sensitive ATPase

activity was estimated and expressed as micromoles or inorganic phosphate released

per mg protein per hour.

Estimation of inorganic phosphate

Theory

The protein free filtrate reacts with acid molybdate solution which forms

phosphomolybdic acid from any phosphate present. The phosphomolybdic acid is

reduced by the addition of 1, 2, 4, aminonapthosulphonic acid reagent to produce a

blue colour whose intensity is proportional to the amount of phosphate present. The

following solutions were prepared for the estimation of inorganic phosphate by the

method describ~d by Fiske and Subbarow (1925)

Assay system for estimation of inorganic phosphate

Molybdate Solution

25 gms of reagent grade ammonium molybdate was dissolved in 200m! of

water. In one litre volumetric flask, 300m! of 1 ON sulphuric acid was taken. The

molybdate solution was added to this and the volume was made up to one litre.

Aminonaphthosulphonic acid reagent (ANSA reagent)

70

0.5 gm of 1,2,4, aminonaphthosulphonic acid was added to 195 ml of 15%

sodium bisulphite solution ·and shaken till the powder dissolved. 5ml of 20% sodium

sulphite solution was added to it. The solution was preserved in a dark bottle in cold.

The assay system, which also contained 1 ml of TCA was centrifuged at

1 OOOxg for 8 minutes. 1 ml of the protein-free supernatant was taken, to which 8.1 ml

of distilled water was added. 0.5ml of ammonium molybdate solution was added and

incubated for 10 min at room temperature. 0.4 ml of ANSA reagent was added and

the colour developed was estimated spectrophotometrically at 660nm. The intensity

was quantified spectrophotometrically at 660nm. KH2P04 in a concentration range of

20J.!g to 1 OOJ.!g was used as a standard.

Specific activity of enzyme

In most preparations, the actual molar concentration of the enzyme is

unknown. Consequently, the amount of enzyme present has been expressed in terms

of its activity as defined by the International Union ofBiochemistry.

Standard unit: One International Unit (IU) of enzyme is an amount of the enzyme

that catalyses the formation of 1 micromole of product per minute under defined

conditions.

The specific activity ofN a-K ATPase was calculated as the difference between

the amount of inorganic phosphate liberated in the presence and absence of ouabain

and expressed as micromoles of inorganic phosphate released per mg protein per

hour.

Comparison of Na-K ATPase activity in microsome and synaptosome

Earlier studies have shown that REM sleep deprivation increases microsomal

71

Na-K ATPase activity. Synaptosome is known to be a relatively purer preparation as

Na-K ATPase is clustered in the synaptic region. Since a comparison of microsomal

and synaptosomal Na-K ATPase activites from the same animal was not available,

a comparative study was done before studying the effect of REM sleep deprivation

and its mechanism of action on synaptosomal Na-K ATPase activity.

As shown in the flowchart (Fig 6), the same brain homogenate was used to

prepare microsome and synaptosome. These were then used for the estimation ofNa

K ATPase activity under various conditions.

Effect qf Lysis

The activities ofNa-K ATPase in synaptosome and microsome were compared

in unlysed and lysed preparations. During the microsome preparation, the final pellet

was suspended in 5mM Tris/ EDTA buffer, pH 7.4. An aliquot was used immediately

· as unlysed microsome. The rest of the suspension was left overnight for lysis at 4oC

and used the next day as lysed microsome.

The synaptosomal band, isolated from the discontinuous sucrose gradient

suspended in isotonic sucrose solution represents the unlysed synaptosome, which are

the pinched off vesicles from the synapse. When these are suspended in distilled

water, centrifuged and the pellet is resuspended in water, it is lysed to synaptosomal

plasma membrane. The specific activities ofNa-K ATPase in the unlysed and the

lysed synaptosomal preparations were compared.

Effect of EDTA

Since calcium is known to inhibit Na-K ATPase (Davis and Vincenzi, 1971)

and calcium levels are found to be altered in synaptosome after REM sleep

72

deprivation (Mallick and Gulyani, 1996), the effect of divalent cation chelator EDTA

on Na-K ATPase activities were studied in the microsome and the synaptosome.

The brains from the control and the experimental group were homogenized in

Sucrose-Tris buffer in the presence and absence of lmM EDTA. The synaptosome

was resuspended in homogenizing buffer in the presence or absence of lmM EDTA.

The microsome was suspended in 5mM Tris or EDTA buffer, pH 7.4 depending on

whether the initial homogenization was done in the absence or presence of EDTA.

Thus, the synaptosome and microsome were prepared in the presence and absence of

EDT A and the enzyme activity was estimated and compared.

Effect of REM sleep deprivation

The effect of REM sleep deprivation on microsomal and synaptosomal Na-K

A TPase was investigated. The experimental rats were kept for REM sleep deprivation

on small platform (6.5cm diameter) for 4 days. This time period was chosen because

it has been shown earlier (from this lab under similar conditions) that atleast 4 days

of REM sleep deprivation was required to increase microsomal Na-K ATPase

activity in whole brain preparations.

The control and the experimental group of rats were decapitated after cervical

dislocation and the brains were removed into chilled buffer within three minutes. The

microsome and the synaptosome were prepared from the same brain homogenate in

the presence and absence of lmM EDTA. The pellet obtained after the 12000xg

centrifugation was used for synaptosome and the supernatant from the centrifugation

was used for microsome preparation (Fig 6). This was done to enable comparison of

microsomal and synaptosomal Na-K ATPase activity from the same animal brain

after REM sleep deprivation. Similarly, the microsomal and synaptosomal preparation

73

PREPARATION OF MICROSOME AND SYNAPTOSOME


6K/ 5'

I PELLET I SUPERNATANT

I PELLET I

: Detailed in ~ Fig 4

SYNAPTOSOME

Fig 6

12K I 20'

SUPERNATANT

Detailed in Fig 5

MICROSOME

from large platform controls were used for the estimation ofNa-K ATPase.

Thus, the activities ofNa-K ATPase were compared in unlysed and lysed

synaptosome prepared from FMC, LPC and REMSD groups in the presence and

absence of EDT A.

Estimation in brain areas

The effect of REM sleep deprivation on synaptosomal Na-K ATPase activity

in different brain areas viz- cerebrum, cerebellum and brain stem were estimated and

compared to that of FMC and LPC groups.

Mechanism of REM sleep deprivation induced increase in Na-K ATPase activity

Since, the synaptosomes showed a much higher specific activity of Na-K

ATPase when compared to the microsome and also showed a increase in Na-K

A TPase activity after REM sleep deprivation, further studies on REM sleep

deprivation were done using synaptosomal preparations only.

In order to investigate the molecular mechanism(s) involved in REM sleep

deprivation induced increase in Na-K ATPase activity in the synaptosome, an in vitro

. system was employed. There are various studies which indicate that the REM sleep

deprivation induced increase in Na-K ATPase activity is mediated by NE. They are

• as fellows:

i)The noradrenergic REM-OFF neurons, which normally cease firing during REM

sleep, do not stop firing during REM sleep deprivation (Mallick et al., 1989). ii) A

decrease in MAO-A, an enzyme that degrades NE has been observed after REM sleep

deprivation (Thakkar and Mallick, 1993). iii) Mild electrical stimulation of

noradrenergic LC neurons, which did not allow the cessation ofNE-ergic REM-OFF

74

neurons, produced an effect similar to that of REM sleep deprivation (Singh and

Mallick, 1996) iv) An increase in turnover of tyrosine hydroxylase- the enzyme

catalysing the first step in NE biosynthesis, has been reported (Sinha et al., 1973,

Basheer et al., 1998). Thus, an increase in NE concentrations in the brain is expected

after REM sleep deprivation. Also, NE is reported to stimulate Na-K ATPase activity

in vitro. Therefore, the effect of REM sleep deprivation can been simulated in vitro

by addition ofNE to the homogenate.

This in vitro model has been used here in this study to investigate the

mechanism ofNE induced increase in Na-K ATPase activity .

. Dose-response of NE

Before investigating the molecular mechanism involved in NE mediated

increase in Na-K ATPase activity, a dose response study was carried out. The dose

response ofNE was done in order to estimate the optimum and effective dose required

for eliciting an increase in Na-K ATPase activity in the unlysed and the lysed

synaptosome. The homogenate was incubated with different doses ofNE at 37oC for

10 minutes before preparation of synaptosome and Na-K ATPase activity was

estimated.The specific activity ofNa-K ATPase was estimated in both the unlysed

and lysed synaptosome.

NE was reported to increase microsomal Na-K ATPase activity (Gulyani and

Mallick, 1995). In this study, the effects ofNE on microsomal and synaptosomal Na

K ATPase activities were compared. The effect ofNE was studied in two ways- i) by

addition to the homogenate before preparation of the microsome and synaptosome

(Fig 7) and ii) by addition to the prepared unlysed, lysed synaptosome and microsome

(Fig 8).

75

·Addition to the homogenate (Fig 7)

EffectofNE

Before preparation of microsome and synaptosome, the homogenate was

divided into two parts. To one part, NE (100 11moles) was added and both the parts

were incubated at 37°C for 10 min. The Na-K ATPase activity was estimated in the

lysed microsome, unlysed and lysed synaptosome prepared from such homogenate.

Effect of NE in the presence of EDTA

Calcium was postulated to modulate NE induced increase in Na-K ATPase

activity. In order to cofirm this, NE stimulation ofNa-K ATPase activity was studied·

in the presence and absence of EDT A. NE was added to a final concentration of

10011M in the brain homogenate which contained or did not contain 1mM EDTA. The

microsome and synaptosome were prepared and the Na-K ATPase activity estimated

in the membrane preparation.

Addition to the microsome and synaptosome (Fig 8)

EffectofNE

Microsome and synaptosome were first prepared from untreated homogenate

which was then treated with NE (l0011M) and the enzyme activity estimated. The

effect of NE was studied by addition to the microsome, unlysed or the lysed

synaptosome by incubating with 10011M NEat 37oC for 10 min, before assaying for

Na-K ATPase activity.

Effect of norepinephrine in the presence of EDTA

Microsome, unlysed and lysed synaptosome, prepared in the presence of 1 mM

76

SCHEMATIC REPRESENTATION OF EXPERIMENTALPROTOCOL

ADDITION TO THE HOMOGENATE

BRAIN HOMOGENATE

PREPARATION OF MICROSOME . AND SYNAPTOSOME

~ ~

PREPARATION OF MICROSOME AND SYNAPTOSOME

ESTIMATION OF Na-K ATPase ACTIVITY

Fig7

SCHEMATIC REPRESENTATION OF EXPERIMENTALPROTOCOL

lr

CNTRL

l m + C) trJ ...., C)

;J> ~

, ,

ADDITION TO THE MICROSOME AND SYNAPTOSOME

UNLYSED SYNAPTOSOME

, ,

, , +PRAZ

+NE +NE

lr lr

BRAIN HOMOGENATE

1 +PROP +NE

,

LYSED SYNAPTOSOME

2 ....., + § ~ trJ

:> C) ....., tr1 ~ tT1

0

, r

MICROSOME

2 .....,

~ ~ + trJ C) ....., tr1 ....,

tTl ;J> 0

, , ,

ESTIMATION OF Na-K ATPase ACTIVITY

Fig 8

EDTA was incubated with 1 OOflM NEat 37oC for 10 mi:1 prior to estimation ofNa-K

A TPase activity.

Effect of adrenoceptor blockers

Earlier studies showed that preincubation of the homogenate with a-adrenergic

blocker -prazosin prevented the NE induced increase in microsomal Na-K ATPase

activity ( Gulyani and Mallick, 1995). Therefore, the effect of adrenergic blockers

prazosin and propranolol was studied on synaptosomal Na-K ATPase activity. Since

NE was found to stimulate synaptosomal Na-K ATPase activity only when added to

the homogenate or to the unlysed synaptosome and only in the absence of EDT A, the

effect of blockers were also studied in the same. As a control, the effect of adrenergic

blockers were also studied on microsomal Na-K ATPase activity.

Addition to the homogenate

The homogenate was treated with NE in the presence of a:-1 or ~-blocker,

Prazosin and Propranolol, respectively, and Na-K ATPase activity was estimated in

the microsome and the synaptosome. In these cases, the homogenate was first

incubated in the presence of 50flM prazosin or propranolol at 37oC for 10 minutes.

The homogenate was then treated with 1 OOflM NE and incubated further for a period

of 10 minutes at 37°C. The microsome and synaptosome were prepared from such

treated homogenate and the Na-K ATPase activity was estimated. The effect of

prazosin alone on Na-K ATPase activity was also studied in the microsomal and

synaptosomal Na-K ATPase (Fig 7).

77

Addition to the synaptosome

The unlysed synaptosome was incubated with prazosin or propranolol for 10

min at 37oC before incubation with NE and estimation ofNa-K ATPase activity.

Since the lysed synaptosome and microsome did not show any increase in Na-K

ATPase activity, when incubated in the presence of NE, the effect of adrenoceptor

blockers was not studied in the lysed synaptosome and microsome (Fig 8).

Effect of EGTA on synaptosomal Na-K ATPase

Since the NE induced increase in Na-K ATPase activity was blocked in the

presence of either EDTA or prazosin, it remained to be studied if the receptor

mediated (prazosin) and the non-receptor mediated (through EDTA) increase in the

enzyme activity culminated in the same pathway. In order to test the hypothesis that

the NE induced increase in Na-K ATPase could be by removal of calcium, the effect

of specific ca++ chelator EGTA [Ethylene glycol-his (b-aminoethyl ether) N,N,N',N'

tetra acetic Acid] on microsomal and synaptosomal Na-K ATPase activity was

studied. The microsome and synaptosome were prepared from either untreated, NE

treated or prazosin+NE treated homogenate. lmM EGTA was added to the assay

system before estimation ofNa-K ATPase activity in the microsome and synaptosome

(Fig 7 & 8).

Since the effects ofREM sleep deprivation and NE treatment on synaptosomal

enzyme activities were similar to that of the changes reported earlier in the

microsomal Na-K ATPase activities, further studies were carried out in the

synaptosomal preparation only.

78

Fluorescence Studies Using Chlorotetracycline

Theory

Tetracycline antibiotics show a propensity for chelation of divalent cations,

which may play a determining role in the bacterostatic properties of the antibiotic.

Caswell and Hutchinson ( 1971) showed that tetracyclines bind preferentially to

cations on membrane surfaces. The chelation of ca++ is reflected in a large increase

in-fluorescence as observed in an apolar solvent.

Chlorotetracycline (CTC) has been used quite extensively to monitor calcium

ions associated with various types of membranes, including mitochondria (Caswell

. and Hutchinson, 1971) and synaptosome (Carvalho, 1978). The studies with NE and

EGTA under different conditions seem to indicate that calcium is likely to be bound

at or near the vicinity ofNa-K ATPase and NE may be causing a stimulation ofNa-K

A TPase activity by removing this bound calcium. Thus, the CTC induced

fluorescence method was used to study the removal of membrane bound calcium in

the presence ofNE and its receptor antagonist.

Method

The synaptosome obtained from the discontinuous sucrose gradient was

washed and resuspended in the homogenizing buffer. Synaptosome containing 0.95

to 1mg protein/ ml was taken to which 12.5J.!M of CTC was added and mixed

thoroughly. The emission spectrum was recorded in the range of 450nm to 600nm to

observe the emission maxima after excitation at 397nm.

The synaptosome was loaded with Ca++ by incubating with lmM CaCl2• The

fluorescence emission spectra was monitored in the range of 450 to 600nm, with an

excitation maxima at 397nm. After determining the amount of fluorescence in the

79

control sample, increasing concentrations ofNE from 0 to 600J.!M was added and the

change in fluorescence spectra was monitored.

The change in fluorescence was then monitored in the absence of CaC12• Since

the Na-K ATPase activity was stimulated by 1 OOJ.!M ofNE, the same concentration

was added, mixed thoroughly and the fluorescence emission was monitored at 522nm

with an excitation maxima at 397nm. Percent changes in the fluorescence levels were

calculated after each addition in all the following cases using a) as the control.

a) Protein + CTC

b) Protein +CTC + NE ( 1 00 J.!M )

c)Protein +CTC+Prazosin (50J..LM) +NE (IOOJ..LM)

d) Protein +CTC +NE (100 J..LM) +EGTA (lmM)

e) Protein +CTC +Prazosin (50J.!M )+NE(100J..LM) +EGTA (1mM)

~GTA was added to the sample containing protein and CTC, which was

pretreated with NE or prazosin+NE, in order to study ifNE is able to chelate most of

theca++ present in the sample. Percent changes in fluorescence were calculated after

each addition and plotted.

Second Messenger Studies

In order to study the molecular mechanism involved in NE induced increase

in Na-K ATPase activity, an aliquot of the rat brain homogenate was incubated with

various stimulators or inhibitors of components of the signal transduction pathway.

The rat brain was homogenized in the Sucrose-Tris buffer, pH 7.4 in the

absence ofEDTA. The homogenate was diluted ten times and 50J..Ll of the sample

containing 20-30J..Lg of protein was used for the estimation ofNa-K ATPase activity.

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Stimulation Studies

These were done to investigate if any of the possible second messengers, when

added to the homogenate in lieu ofNE, could mimic the action ofNE. In such a case,

that second messenger could be one of the components of signal transduction pathway

initiated at the adrenoceptor and leading to the stimulation ofNa-K ATPase activity.

The homogenate was treated with either NE (IOOJ.!M), cAMP (5J.!M), adenylate

cyclase activator-forskolin (5J.!M), a ca++ ionophore-A23187 (6J.!M) or calmodulin

activator (2U) for 10 min at 37oC before estimating the Na-K ATPase activity.

Inhibition Studies

The rationale of these experiments was that if the pathway from the

adrenoceptor to the enzyme was blocked at any of the steps by preincubation with

these inhibitors, no increase in Na-K ATPase activity would be observed when

stimulated with NE. If the pre-incubation with any of these inhibitors was unable to

block the NE induced increase in Na-K ATPase activity, it could be inferred that the

molecule in question may not be playing an important role in eliciting the increase,

although it may still be playing a modulatory role.

In order to study the second messengers involved in the pathway leading to an

increase in Na-K ATPase activity, the homogenate was preincubated for 10 min at

37oC in the presence of either o:-1 antagonist- prazosin (50J.!M) a-lA antagonist

WB4101 (IOJ.!M), IP3 antagonist-heparin (lU), Calmodulin antagonist-W13 (5J.!M)

and Calcineurin inhibitor-Cyclosporin A (2J..LM) before incubation with NE (lOOJ.!M).

The Na-K ATPase activities were estimated in such treated homogenates.

KINETICS OF Na-K ATPase

Since REM sleep deprivation was found to increase synaptosomal Na-K

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A TPase activites, studies were carried out to investigate the change in kinetic

parameters ofNa-K ATPase in control and experimental groups. The activities ofNa

K ATPase was estimated in the presence of different concentrations of the substrate

A TP, using the coupled assay method in different groups.

In this, three enzymatic reactions are operative in the assay:

Na-K ATPase

ATP +H20 ----------------->Pi+ AqB)

Pyruvate kinase

ADP +PEP-----------------> ATP + Py (2) Lactate Dehydrogenase

Py + NADH + H+ ________ --------------->La+ NAD+ (3)

ADP: Adenosine diphosphate; PEP: Phosphoenolpyruvate; Py: Pyruvate; NAD+:

Nicotinamide adenine dinucleotide; NADH: Reduced form ofNAD; La: Lactate.

Reaction (1) requires Mg++ ; reaction (2) requires Mg++ and K+. During the assay,

which is started by the addition of Na-K ATPase, all the reactions, though are

reversible tend to proceed to the right. The rate of reaction (3) is determined as the

rate of change of absorbance at 340nm (dA340/dt) under conditions where this rate

becomes constant and equal to the rates of the reactions (1) and (2). The steady state

value of dA340/dt measures the uninhibited rate of reaction. The difference in

extinction coefficient at 340nm between NADH (+H+) and NAD is 6.22x103 cm·1 M-1•

Method:

The specific activity ofNa-K ATPase was estimated using the coupled assay

method by Fritz and Hamrick (1966) as described above using substrate

concentrations in the range between 0.1M to 4mM. A 1 ml reaction mixture contained

SmM MgC12, 100mM NaCl, 20mM KCl, 135mM lmidazole-Cl buffer, pH 7.5,

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0.2mM NADH, 0.5mM phosphoenolpyruvate, 0.02% bovine serum albumin, 9 units

of pyruvate kinase and 12 units oflactic acid dehydrogenase. A 25J..Ll synaptosomal

preparation was used with a protein content of20-30J..Lg. Absorbance changes in the

reaction mixture were measured at 340nm using a Hitachi UV2000 spectrophotometer

with the temperature controlled at 37°C. The decrease in optical density at 340nm

over a period of 10 minutes was used in calculating the specific activity expressed as

micromoles Pi per mg protein per hour.

The specific activities obtained with different substrate concentrations were

plotted against the substrate concentrations. The reciprocal of specific activities and

substrate concentrations were plotted as LineWeaver Burke plot.

Kinetics ofNa-K ATPase after REM sleep deprivation

The specific activities of Na-K ATPase were determined using different

substrate concentrations in the lysed synaptosome from FMC, LPC and REMSD

groups. The apparent Km and V max of the enzyme were calculated from the x and the

y intercepts.

. Kinetics ofNa-K ATPase after NE treatment

The REM sleep deprivation induced increase in Na-K ATPase activity was

postulated to be mediated by NE (Gulyani and Mallick, 1995). Thus, the effect ofNE

on kinetic parameters was studied. The homogenate was treated with NE (lOOJ..LM)

and the enzyme activities estimated in the lysed synaptosome using different substrate

concentrations.

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Kinetics ofNa-K ATPase after Ca++ treatment

ca++ was proposed to play a role in mediating REM sleep deprivation induced

increase in enzyme activity (Mallick and Gulyani, 1996). A dose response study of

ca++ ~as first done to study its effect on Na-K ATPase activity. The effect ofca++ on

kinetic parameters ofNa-K ATPase was studied in the lysed synaptosome.

Estimation of lipid peroxidation

There are different theories put forward by various authors to explain the

mechanism of noradrenergic stimulation of Na-K ATPase activity. Since lipid

peroxidation is known to inhibit N a-K ATPase activity, NE may stimulate the enzyme

by inhibiting lipid peroxidation (Schaefer et al., 1972). These authors have shown a

decrease in lipid peroxidation concomitant with an increase in Na-K ATPase activity

in vitro, thus proposing that NE increased Na-K ATPase activity by inhibiting lipid

peroxidation. Since, the REM sleep deprivation induced increase in Na-K ATPase

activity has been proposed to be mediated by NE, the lipid peroxidation levels were

compared in the FMC, REMSD and LPC groups to study if change in lipid

peroxidation by NE may play a role in the stimulation ofNa-K ATPase activity.

Theory

Lipid peroxidation is a complex process involving the formation and

propagation of lipid radicals, the uptake of oxygen, a rearrangement of the double

bonds in the unsaturated lipids and eventual destruction of membrane lipids. The

destruction in membrane lipids produces a variety of breakdown products, including

ketones, alcohols, aldehydes and ethers. Since the biological membranes are rich in

unsaturated fatty acids and bathed in an oxygen-rich metal containing fluid, the

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membrane lipids are very susceptible to peroxidative attack. Lipid peroxidation

usually begins with the abstraction of a hydrogen atom from an unsaturated fatty acid,

resulting in the formation of a lipid radical (Pryor and Stanley, 1975). The

. rearrangement of the double bonds results in the formation of conjugated dienes.

Attack of the conjugated dienes by molecular oxygen produces a lipid peroxy radical,

which can either abstract a hydrogen atom from an adjacent lipid to form a lipid

hydroperoxide, or form a lipid endoperoxide. The formation of lipid endoperoxides

in unsaturated fatty acids containing at least 3 methylene interrupted double bonds can

lead to the formation of malondialdehyde as a breakdown product.

Thus, malondialdehyde (MDA), formed from the breakdown of

polyunsaturated fatty acids serves as a convenient index. for determining the extent of

the peroxidation system. MDA has been identified as the product oflipid peroxidation

that reacts with thiobarbituric acid to give a red species absorbing at 535nm. This

method for lipid peroxidation measurement was described by Rehncrona et al (1980).

Method

The whole brains or the brain areas were homogenized in IOmM Tris-0.15 M

KCl buffer, pH7 .4. Initially, lipid peroxidation levels were estimated in the

supernatant obtained after centrifuging the brain homogenate at I O,OOOxg for 20 min.

After standardization, the same were done in lysed synaptosomes.

About 0.4mg of protein sample from the soluble fraction or the synaptosome

was diluted to 2 ml with IOmMTris buffer containing 0.15M KCl solution at pH 7.4.

0.5 ml of30% TCA solution and 0.5ml of52mM TBA solution were added, vortexed

and incubated in a waterbath at 80oC for 45 min. The tubes were cooled on ice and

centrifuged for 10 min at 6000xg. The supernatant obtained was read

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spectrophotmetrically against blank at 531.8 nm. A molar extinction coefficient of

1.52xl05 mol- 1 cm· 1 was used.

The amount of lipid peroxidation was calculated as nmoles ofMDA produced

per mg protein.

Endogenous lipid peroxidation

The brain areas- cerebrum, cerebellum and brainstem from FMC, LPC and

REMSD rat brains dissected out and homogenized in Tris-KCl buffer seperately. The

amount of MDA was estimated in the soluble fractions of the brain areas of the

experimental and control rat groups.

Time course of lipid peroxidation

The peroxidation of the samples obtained from FMC, LPC and REMSD rat

brains was induced by incubating the protein in Tris-KCl buffer at 37oC for different

time intervals. The reaction was stopped at 0', 5', 10', 20', 30', 45' and 60' by the

addition of0.5ml TCA and 0.5ml TBA. The tubes were incubated in a waterbath for

45 min and proceeded for MDA estimation as mentioned above. The amount of MDA

produced was calculated and compared between the different experimental and

control groups.

Comparison of lipid peroxidation after REM sleep deprivation and NE treatment

Since an increase in synaptosomal Na-K ATPase activity was observed after

REM sleep deprivation and after NE treatment, a comparison of synaptosomal lipid

peroxidation levels was done after these treatments.

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3.3 STATISTICAL ANALYSIS OF DATA

A statistical hypothesis is an assumption made about a parameter in a

population. Since, it is impossible to test this in the entire population, a sample of the

population is used for the study. In most cases, only estimates of the parameters

obtained from random samples are available and the assumptions must be tested using

these estimated. These studies are called the test of significance.

There are two kinds of hypotheses: the Null hypothesis (H0), which is a

hypothesis of no differences. It is usually formulated for the purpose of being

rejected. If it is rejected, the alternative hypothesis (H1) may be accepted. The

alternative hypothesis is the operational statement of the experimenter's research

hypothesis.

If H0 is to be rejected in favor of HI> a statistical test should yield a value

whose associated probability of occurrence under H0 is equal to less than some small

probability symbolized as ex or P. This small probability is called the level of

~ignficance. Common values of ex are 0.05 and 0.0 1. In other words, the statistical test

involves determining the P value, which can be defined as the probability that one is

incorrect in stating that the two means are different.

ANOVA or Analysis Of Variance has been used to determine the significance

level in the various experiments. The assumptions associated with the statistical

model underlying this test are: that the observations are independently drawn from

normally distributed populations; that the populations all have the same variance. This

test performs simple analysis of variance (anova) to test the hypothesis that means

from two or more samples are equal (drawn from populations with the same mean).

This technique expands on the tests for two means, such as the t-test.

If the groups are random samples from the same population, the two variances,

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within and between, are unbiased estimates of the same population variance.

Significance of the difference of the two types is tested by the use ofF test.

mean-square for between groups F

mean-square for within groups

where the mean-squares are obtained by dividing each of sum of squares by its

respective number of degrees of freedom.

The sum of squares between the various groups can be found by taking the

mean of each group, getting its deviation from the total mean, squaring this deviation,

and then multiplying each ofthese by the number of individuals in each group.

The within sum of squares for each group can be found by

n where X is the mean of each group and n is the number of samples.

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the details of the materials and methods used in this...

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