. . ........ ''

M A T E R I A L S A N D M E T H 0 D S

66

MATERIALS AND METHODS

EXPERIMENTAL ANIMALS

Swiss albino mice, obtained from the experimental animal

facility of AIIMS, New Delhi and maintained with random breeding

in our experimental animal facility at JNU, were used in this

study. They were caged in semitransparent polypropylene cages

(size 8" x 12" x 5") with covers made of stainless-steel grill.

Approximately 20 mice were housed in a cage. The bedding material,

rice-husk, was changed twice a week. Food and water were provided

ad libitum. Food is mainly the rat food pellets fr.om Hindustan

Lever Ltd., Delhi and occasionally leafy-vegetables like cabbage

or palak were also included. The temperature of the animal house

was maintained at roughly around 25°C. The rooms remained lighted

during the day-time. Mice of approximately 12-16 weeks and

6-8 weeks were used for obtaining spleen and thymus lymphocytes

respectively.

CHEivliCALS AND MATERIALS

All the chemicals used were of analytical grade and were

obtained either from B.D.H., Bombay or from Sigma Chemical Co.,

U.S.A. RPMI 1640 medium, Fetal Calf Serum (FCS) and Poke Weed

Mitogen (PWM) were from Grand Island Biological Co. (GIBCO) ,

U.S.A. FCS from Centron Research Labs., Bombay or from Sera-Labs.,

U.K. was also used for removing glass-adherent cells. Phytohema­

gglutinin, type M (PHA-M) was from Difco, U.K. Concanavalin A

(Con.A) was from Sigma, U.S.A. Calcium ionophore was the gift

67

from M/s. Eli Lily Co~, U.S.A. Arsenazo III was from LOBA-CHEM,

Bombay. Membrane filters were from Max-Flow, Bombay and glass

fibre filters, type GF/C were from Whatman co:, U.K. Micro-test

plates (flat bottom, 96 well) and the detergent for cell culture

glass-ware c:teaning were from Luxbro, Bombay. Tritium labelled

thymidine ( 3H-Thymidine) and Calciurn-45 isotope were obtained

from B.A.R.C., Bombay.

Methyl glyoxal was purified by distillation at 75~2°C.

Arsenazo III was purified by the column chromatography as

suggested by Kendrick (418). Monodansyl derivative of diamino­

butane was prepared by the conventional dansyl reaction (601).

GLASS-WARE

All the glassware used were thoroughly clean. Chromic

aci.d was never used. Instead 50% HN03

(for general glassware),

80% H2so4

plus 5% HN03

in distilled water (for cell culture

glassware) or80% HN03

plus 20% HCl (for glass beads) was

used for the acid dip and a detergent for cell culture from

Luxbro (India) was used for detergent wash. A thorough brushing

and plenty of rinsing with tap water as well as with distilled

water for the final cleaning was always given the prime impor­

tance. Glass beads were cleaned by immersing in a mixture of

80% Cone. HN0 3 and 20% Cone. HCl for one day, followed by washing

with distilled water. They were then immersed in alkaline

methanol for 1 hr. Then they were extensively .rinsed with

distilled water, dried and siliconized. All the glassware thae

68

were used/employed in the spectrophotometric assays of calcium

fluxes were further processed for removing the adsorbed metals

by immersing the clean glassware in 1% EDTA solution for one

day followed by thorough rinsing with 4-time distilled water to

remove EDTA and then dried.

STERILIZATION

All the materials were sterilized in oven (250°C, overnight)

wherever possible or by autoclaving (15 psi, 30') wherever

necessary. All the other materials which cannot be sterilized

by the above methods were sterilized by dipping fn 80-85% ethyl

alcohol, followed by thorough rinsing with the autoclaved

distilled water and subsequent drying at RT under sterile condi­

tions, using horizontal laminar flow chamber.

CULTURE MEDIUM AND OTHER SOLUTIONS

RPMI medium and the solutions that come in contact with

the lymphocytes were prepared using 4-times distilled water having

a specific resistance of about 5 lakh ohms. RPMI medium was

prepared from the powdered medium and supplemented with 2mM

L-glutamine 1 5, mM sodium pyruvate 1 50 )4M 2-mercapto-ethanol,

100 U/ml pencillin G, 100 pg/ml streptomycin sulphate, 24mM

sodium bicarbonate and 50 mM HEPES and sufficient amounts 1N

NaOH to bring the final pH of the medium to 7.2. The medium was

sterilized by membrane filteration using 0.22pm pore-size Max­

Flow filters and stored in well-stoppered bottles at 4-5°c in a

refrigerator. FCS was added to the medium upto 10% just before

69

use. Stock solutions of Con.A, PHA-M, PWM drugs and other

chemicals were prepared in 25mM HEPES-HBSS, were sterilized by

membrane filteration as above and were stored at -20°C. Proper

dilutions of these solutions were done in sterile solution of

25mM HEPES-HBSS at the time of experiment.

Balanced salt solutions and other solutions that were used

in cell preparation or in short-time incubations were prepared

from the original ingredients and were sterilized in the same

way as above, but stored at RT.

GENERAL PRECAUTIONS

Every possible care was taken to ensure the sterile condi­

tions during the cell preparation, incubations and culture of

the cells. Cell suspensions were exposed only when they were

in laminar flow chamber. Dissections were carried out in a

closed room of considerable sterility. After .. killing, the mice

were thoroughly swabbed to ensure sterility with 85% ethanol.

The procedures for isolation and purification of lymphocytes

from the lymphoid organs and their culturing methods used in the

present study were developed with some modifications from the

original procedures described by various authors (297, 374, 561,

57 8 1 6 0 8 1 6 1 9 ~ 62 1 1 6 9 3 1 7 0 1 ) o

PREPARATION OF SPLEEN CELL SUSPENSION

The mice were killed by cervical dislocation, spleens were

carefully diseected out, cleared off any fatty tissue remained

70

with them and suspended in HBSS. The fine spleen cell suspension

was obtained by releasing the cells into HBSS by gently teasing

and pressing the spleens against a fine stainless· steel wire-mesh

immersed in HBSS with the help of a syringe barrel and forceps,

then aspirating repeatedly through the Pasteur pipette while

avoiding air bubbles, by twice washing the cell suspension, after

the tissue fragments and cell clumps settled down, with HBSS by

centrifugation at 2000 rpm in a Remi (R8C) centrifuge for 10

minutes and finally passing the cell suspension through a

syringe column loosely packed with cotton wool.

PREPARATION OF THYMUS CELL SUSPENSION

The mice were killed by 100% co2 anaesthesia, thymuses were

gently removed after exposing them by opening the thoracic cavity

by a mid-sternal incision and placed in HBSS. The fine thymus

cell suspension was obtained by the same procedure as described

above.

REMOVAL OF ADHERENT CELLS

The lymphoid cell suspension in HBSS was brought to 10%

FCS and was warmed to 37°C. They were then slowly passed at 37°C·,,

through a previously warmed glass-column (2.5 x 40 em) having

HBSS plus 10% FCS and filled with siliconized, clean glass beads

of 100pm average diameter upto 30 em mark. The column was .. -------

further washed with a column-volume of HBSS plus 10% FCS at 37°c

and the unadhered cells were collected from the column outlet.

71

The glass-adherent cells were removed by washing the column

with 10 columnvolumes of Calcium and Magnesium-free HBSS

containing 0.02% EDTA and were discarded.

The non-adherent cell suspensions were pelleted out by

centrifugation and suspended in a minimum volume of PBS.

REMOVAL OF RED CELLS FROM NON-ADHERENT CELL SUSPENSIONS

The red cells in the above non-adherent cell suspension

were lysed by adding 10 volumes of PBS diluted 10-fold with

distilled water and bringing the resulting suspension to normal

osmotic strength by adding PBS after one minute'. The lysed red

cells were freed off by passing through a syringe column loosely

packed with cotton wool. The effluent was washed by centrifuga­

tion and finally suspended in HBSS. If the lysis was not complete

as rarely it occurs, the process was repeated.

REMOVAL OF RED CELLS FROM LYMPHOID CELL SUSPENSIONS

The same above procedure was followed for cell suspensions

obtained from spleen or thymus whenever the removal of adherent

cells was not required.

Lymphocytes that were freed of adherent cells were used for

all the assays except for the assay of mitogenesis (3H-Thymidine

incorporation assay).

ESTIMATION OF CELL DENSITY AND CELL VIABILITY

C@ll density and cell viability were measured with the hemo­

cytometer using Leitz Aristophot Microscope under phase contrast

72

mode according to the procedure of Absher (2) using nigrosin

as the vital dye. The cells were used either immediately or

stored for not more than 8 hrs. Whenever storage for more

than 2 hrs. is required, the cells were suspended in RPMI 1640

(complete) medium and stored at 5°C. Before use the cells were

pre-warmed at room temperature for 2 hrs., washed by centrifu­

gation and finally suspended in either RPMI 1640 (complete)

medium supplemented with 10% FCS or 25 mM HEPES-HBSS. The cell

suspensions were again subjected to the cell viability and cell

density measurements as described above and were then used for

the experiments. Only those cell preparations hav.ing more than

90% viable cells -were used. In most of the preparations, the cell

viability was above 95%.

EXPERIMENTAL PLAN

To assess the role of calmodulin in the lymphocyte activa­

tion trifluoperazine (TFP), chlorpromazine (CPZ) and lidocaine

(LID) were used. Different concentrations of these drugs ranging

from 0.1)UM to 5 mM were used to see the kinetics of their

effects. When the kinetics of mitogens' effects were studied,

10 ,.uM of TFP, 50 )UM of CPZ and 100 ,.uM of LID which approximately

represent their Ic50 values on the calmodulin activity, were

used. To verify the role of calcium, calcium ionophore A23187

and calcium-specific divalent metal ion chelator, ethylene

glycol bis <p-aminoethyl ether) N,N,N',N' -tetra acetic acid

(EGTA) were used. Chlortetracycline (CTL) was also used as a

presumed calcium ionophore since it was generally used as a

STRUCTURES OF THE CHEMICALS USED IN THE PRESENT STUDY

'Irifl uoperazine

Chlorpromazine

Lidocaine

A23187

Chlortetracycline

EGTA

CH-N j I

H CooH

Cyclic AMP

8-Brorno cyclic AMP

Dibutyryl cyclic AMP

Isobutyl methyl xanthine

Papaverine

Theophylline

Cyclic C::MP

Dibutyryl Cyclic GMP

Acetylcholine

Carbarnylcholine

MNNG

Sodium nitroprusside

Levarnisole

I OH

- ~-:OifHfH.) 0

NH tl

CH~- N-c -NHWO ¥ I ,.

No

73

calcium specific fluorescent probe in the lipid phase which

may possibly a sufficient indication for the ionophoric activity

of.a tetracyclic molecule having three hydroxyl and two carbonyl

oxygen groups on one side for holding ca2+ ions with considerable

affinity and the hydrophobic nature of the opposite side of the

molecule making it easy to traverse/diffuse across the lipid

bilayer. 10 nM of A23187, 100 nM of CTL and 5 mM of EGTA were

used in the present study. To assess the role of cAMP; 0.1 and

1 pM cone. of cAMP, 10 and 100 nM cone. of its derivatives namely

8-bromo-cAMP and dibutyryl-cAMP and 1 and 10 rnM cone. of papave­

rine, theophylline and isobutyl methyl xanthine (IBMX) were used.

To assess the role of cGMP;0.1 and 1 JIM cone. of cGMP, 10 and 100

nM cone. of its derivative namely dibutyryl-cGMP and the pharma­

cological agents known to increase the cGMP levels namely acetyl­

choline at 1 and 10 pM cone., carbamylcholine at 0. 1 and 1 ,uM ·

cone., sodium nitroprusside and N-methyl-N'-nitro-N-nitrosa-

guanidine at 10 and 100 pM cone. and levamisole at 10, 100 and

1000)1M cone. were used.

ASSAY OF MITOGENESIS (3H-THYMIDINE INCORPORATION ASSAY)

Lymphocytes in triplicate were cultured at 37°C in 96 -

well, flat bottomed microtest plates at 5 x 106 cells/ml in RPMI

1640 (complete) medium supplemented with 10% FCS eithe.r alone or

with the mitogens with or without the drugs and other substances.

The plates were kept in air-tight dessicators gassed with 5% co2

in air. The culture time was 72 hrs. Each well received 1 JICi of

74

3H-thymidine in 10 pl of RPMI 1640 (complete) medium during the

last 24 hrs. i.e. at 48 hrs. of the culture time. After 72 hrs.

of culture, the plates were removed from the incubator, kept

on ice and immediately further processed for collecting the tri­

chloroacetic acid (TCA) insoluble material on the glass fibre

(GF/C) filters. Cell suspension from each well of the plate

was collected on the filter that was kept on the sintered glass

disc of the filteration apparatus and washed twice with 5 ml

of PBS, twice with 5 ml of 10% TCA, twice with 5 ml of 85% ethanol

and finally once with 5 ml of chloroform . The. filter was

then dried under infra~red lamp (hot lamp), transferred to the

glass scintillation vial containing 10 ml of scintillation fluid

(10 gm PPO, 0.625 gm POPOP per 2.5 litre of sulpher-free tolune)

and counted for radioactivity in LKB - Bromma Liquid Scintilla-

tion Counter (1217 RACK BETA).

In the experiments involving the pulse-exposure of Con.A

to the cultures, ~methyl mannoside in RPMI 1640 (complete)

medium was added to the cultures to a final cone. of 0.1M and

the cultures were further incubated and processed for the assay

essentially as above.

ASSAY OF CALCIUM-45-UPTAKE

200Jll of cell suspension (2.5 x 107

cells/ml) was taken

in a glass tube and pre-incubated for 10 minutes at 37°C. The

uptake reaction was started by quickly adding 25JU1 of solution A

(25 mM HEPES-HBSS) or solution A containing sufficient cone. of

75

mitogen or drug or both to give the necessary final concentrations

and 25 pl of Ca-45 solution ( 1-2 pCi per tube) in that order and

tne contents·- were gently mixed immediately. The reaction was

terminated by filtering 200 pl of the contents of the tube onto

a membrane filter (0.45 pm pore size, 2.5 ern diameter) that was

placed on the sintered glass disc of the filteration assembly

attached to vacuum pump and prewashed with 5 rnl of 50 rnM Cac1 2

solution. Three washings of 5 rnl each of solutions A, were

followed to wash the cells on the filter immediately. Then the

filter was taken out and dried under the hot lamp. The background

or non-specific binding of the isotope to the filter was obtained

by repeating the above procedure but without cells. The radio-

activity on the filter was measured similarly as in the case of

3H-thyrnidine incorporation assay. The Ca-45 uptake by the cells

was expressed as CPM after substracting the CPM due to the non-

specific binding of Ca-45 to the filter.

ASSAY OF CALCIUM NET FLUXES

These assays were done according to the method of Scarpa

et. al., {593) using murexide and arsenazo III as calcium sensitive

dyes and Schirnad~u UV-3000 dual wavelength single beam spectro­

photometer. The assay mixture contained 140 rnM NaCl, 5 rnM KCl,

1 rng/rnl glucose. 25 pM CaC1 2 plus 25 JIM arsenazo III or 1 rnM

CaCl2 plus 100JlM murexide, 50 rnM HEPES (pH 7.2) and cells at

7 the cone. of 2.5 x 10 cells/rnl. The wavelength differentials

of 542-470 and 525-600nrn were used for murexide and arsenazo III

respectively. After the initial stabilization of the trace, 10Jtll

76

of either the reaction mixture/assay mixture without cells or

the same containing either Con.A, drugs or both was added and

2+ the continuous changes in the extracellular Ca levels were

recorded as function of time at 37°C.

SAMPLE PREPARATION FOR THE ENZYME ASSAYS

Non-adherent cells were suspended in 25mM HEPES-HBSS at the

cone. of 5 x 10 7 cells/ml in test tubes and were incubated at

37°C in a serological water bath for 1 hr. (unless otherwise

mentioned) with or without mitogens, drugs and ~ther substances.

Later they were centrifuged for 10 minutes at 2000 rpm in a Remi

Centrifuge and the cell pellet was washed once with 25 mM HEPES­

HBSS containing 0. 1 M cl-methyl mannoside and then once with

25 mM HEPES-HBSS. Finally the cell pellet was suspended in 1 ml

of the buffer solution that was used for the enzyme assay and

was homogenized using Potter Elvehjem type homogenizer fitted with

teflon plunger. All the homogenized samples were stored at -20°C

till the enzyme assay.

ASSAY OF Ca2

+ -ACTIVATED NEUTRAL PROTEASE

This enzyme catalyzes the proteolysis in the presence of

calcium ions. The enzyme was assayed according to the method of

Kawashima et al., (416) .• The reaction mixture (0.5 ml) contained

0.24% alkali-denatured casein, 28 mM 2-ME, 6mM cac1 2 and 0.1 M

sodium glycerophosphate-HCl (pH 7.5) and the sample. The reaction

was started by the addition of the sample and was stopped by the

77

addition of 0.5 ml of 10% TCA after incubating for 15 minutes

0 at 37 C and centrifuged at 3000 rpm in a Remi centrifuge for 10

minutes~ The absorbance of the supernatant was measured in

Schimadzu UV-3000 spectrophotometer against a blank that was

prepared for each sample in the same way except that 10 rnM EGTA

was present in the reaction mixture in which cac1 2 was omitted.

Enzyme activity was expressed as Units; one unit being equal

to 0.001 A/108cells/min. under the standard assay conditions.

ASSAY OF TRANSGLUTAMINASE

This enzyme catalyzes ca 2+ -dependent acyl transfer

reactions physiologically leading to the formation of e<Y-glutamyl)

lysyl cross-links between proteins, as shown below:

0 . ,., Pro te1n - R

1-c ·- NH

2 + Ha,N - R

2 - Protein >

0 It

Protein - R1-c - HN-R2-Protein + NH3

This enzyme can be assayed biochemically/fluorimetrically

as the incorporation of Dansyl compounds having primary amino

group into some proteins like ~-casein catalyzed by it in the

following reaction.

NH 2 Protein - ~lu + H N-R 2

NH-R

' ----~) Protein - Glu +

It was assayed according to the method of Lorand et al.,

(460, 461) with some modifications. The reaction mixture (100 pl)

contained the final concentrations of 40pM Dansyl-butylamine,

20 p~:M~casein, 25 pi!! cac12 , 6 rnM Nael, 50 rnM HEPES (pH 7. 5) and

78

the sample. The reaction was started by adding the sample and

the reaction time was 60 minutes at 37°C and was terminated by

the addition of TCA to 7% final concentration. The precipitate

was dissolved in 5 M urea + 0.5% SDS, pH 8.0 and the protein

bound fluorescence was measured at 520 nm by exiting at 360 nm.

The blank without enzyme was also used. The activity was expre-

ssed as RFE (Relative Fluorescence Enhancement) calculated as

below:

RFE = (Fluorescence of sample - Fluorescence of blank) I

(Fluorescence of blank)

ASSAY OF GLYOXALSE I

This enzyme catalyzes the following reaction:

Methyl glyoxal + GSH S-lactoyl GSH

This enzyme was assayed at 37°C according to the method

of Alexander and Boyer (13) with some modification. The reaction

mixture consists (0.1 ml) of 10 mM methylglyoxal1 2 mM GSH,

50 mM HEPES (pH 7.4) and the sample. The reaction was started by

0 adding the sample, to the reaction mixture at 37 c. Reaction was

terminated by diluting the contents of the reaction mixture with

300 volumes of 66 mM semicarbazide HCl in the phosphate buffer

and the decrease in the optical density at 282 nm was measured

against the semicarbazide blank using Schimadzu UV-240 or UV-260

spectrophotometer or Carl-Zeiss Zena spectrophotometer. The

millimolar exinction coefficient of 32 was used for calculating

79

the enzyme activity which was expressed as n.moles of methylgly­

oxal reacted per 10 6 cells per hr.

ASSAY OF METHYLGLYOXAL NEUTRALIZATION

This was done using the same principle of glyoxalase I assay

i.e. the formation of methylglyoxal disemicarbazone adduct.

Methyl glyoxal was added to the cultures of lymphocytes in

HEPES-HBSS at specified times and incubated further for 60 minu-

tes after which the culture was diluted 600-fold with semicarbazide

HCl in the phosphate buffer and was shaken vigorously in the

water bath shaker, The decrease in the O.D. at 282 nm was measured

6 and expressed as n.moles of methylglyoxal consumed per· 10 cells

per hr., which was calculated using the millimolar extinction

coefficient of 32 for methylglyoxal disemicarbazone adduct.