Acta histochem . Bd. 62, S. 12 - 25 (1978)

Cattedra di Chi mica Biologica, ]'aeoIta di Farmaeia, Universita, Milano, Cattedra di I stologia, FacoIta di Seienze, Universita, Modena,

I stituto di Biologia Generale, Facolta di Medicina e Chirurgia, Universita, Milano, ed Istituto di Anatomia Comparata, Universita, Pavia, Italy

Biochemical and histochemical features of human cultured cells (EUE) adapted to hypertonic medium

By LOBENZO BOLOGNANI, ANNA M. BOLOGNANI FANT1N, ANNA M. FUHRMAN CONTI, MARIA V. GERVASO and MARIA F. OMODEO SALE

With 7 figures

(Received September 15, 1977)

Summary

EUE cells from a human heteroploid line cultured in hypertonic medium (0. 274 M NaCI) modify their lipid pattern: sulfolipid con centration reaches 86 to 90 Ilg/mg protein whilst it ranges between 19 to 32 Ilg/mg in cells cultured in isotonic medium.

Ganglioside concentration reaches 2.6 nmoles of sialic acid/mg protein (after 75 days) and 13 (after 85 days) in hypertonic saline medium. Whilst it is 0.5 in isotonic m edium. Phospholipid concentration does not show any similar change.

Cytoenzymatic analysis reveals that dehydrogenases (lactate, G·6·P dehydrogenases, tetra· bydrofolate reductase and NADH diaphorase) appear strongly enhanced in cells grown on hyper. tonic m edium. On the contrary higher acid phosphatase and ATPase activity was demonstrable in cells grown on isotonic medium.

These results are similar (except for ATPase activi t y) to those observed in salt secreting glands involved in strong osmotic work. The results are discussed in relation to the problem of energy supply in cells performing osmotic work.

Introduction

Biochemical and cytological observations dealing with structures involved in os­motic work are abundant in the literature. It is generally accepted (BONTING 1970; SCHMIDT-NIELSEN 1965) that active ion transport acts as a driving force for fluid transport in several organs (intestine, kidney, urinary and gall bladder, choroid plexus, gills and salt glands) . In these cases a membrane-bound ATPase (Na+ and K+ ac­tivated) is very important as a regulatory device ; a high level of oxoreductases is generally considered to be required in view of the metabolic pathway involved in the coupled synthesis of ATP.

Histological and biochemical studies on salt glands have demonstrated several structural and metabolic capabilities, which may be considered as prerequisites for

Biochemi cal and histochemical features 13

active ion transport (ELLIS and ABEL 1964 ; GERZELI 1967, 1968; KARLSSON 1968,

1971; BOLOGNANI 1970, 1971, 1976). Large amounts of sulphatides are present in

these tissues; the sulpholi pid concentration increases sharply in salt-secreting glands

when submitted to osmotic work (KARLSSON 1968; BOLOGNANI 1970, 1976; GERZELI et a!. 1971, 1976).

More recently our group has been interested in characterizing variants for osmotic resistance in mammalian cultured cells. One particular aim of our investigation was to establish whether EVE cells from a human heteroploid line cultured in hypertonic medium (0.274 M NaCI) modify their lipid pattern and hif>toenzymatic features , as is the case with salt glands. A similar response to salinity, i.e. an increment of sulpho­lipids, is demonstrable in EVE celh cultured in hypertonic medium (BOLOGNANI and CONTI 1974; TORRE et a1. 1974; BOr.oUNANI et al. 1975, 1976); sulphatide concentra­tion reaches 120 fig/mg protein, whereas it ranges between 13 and 39 fig/mg protein in cells cultured in an isotonic medium (0.137 M NaCl). Phospholipid concentration does not show any similar change. The purpose of the present investigation was to confirm our previous data concerning sulpho- and phospholipids and to establish whether any increase in other polar lipids, i.e. gangliosides, also occurs in the cultured cells exposed to hypertonic saline solution. Cytological and histochemical analyses have been also carried out in parallel on EVE cells cultured on hypertonic medium and isotonic medium with a view to demonstrating that there is an increase in polar lipids and histo-enzymatic changes, as these have been considered morphochemical pre­requisites for supporting osmotic work (BONTING 1970; BOLOGNANI et al. 1976; GER­ZELI et al. 1976).

A. Materials and Methods

1. Cell cultures

All the experiments were carried out on cells from t.he EUE line maintained in EAGLE'S medium,

supplemented with 10 % calf serum. The hypertonic medium was obtained by bringing the NaCl concentration in the HANK'S saline from 0.137 M, i.e. the isotonic value, to 0.356 M. Both plating

efficiency and growth rate were evaluated on cells grown in hypertonic medium as compared with controls.

2. Biochemical analysis

a) Extraction of sulpho- a nd phospholipids

The sediment of trypsinized cell s, wltsj)f'd three times in HANK'S solution and spun down at

300 g X 5 min was homogenized in 2 volumes of methanol. Homogenization was repeated after

adding I volume of chloroform . Pa,·tition was t.hen performed by the procedure of BERRA et al. (1974) .

b) Extraction of gangliosides

The sediment of trypsinized ce lls washed in HA"'K'S solution and spun down as before was homogenized in phoHphate buffer (0.01 M pH = 6.8). Eight volumes of tetrahydrofuran were added to ea ch volume of homogenate and extraction was performed in accor<ianee ,,·ith the fol­lowing schen1f':

14 L. BOLOGNANI et 81.

Centrifugation

I .} t

Sediment Supernatant

t (aqueous phase) washed with:

1 ml buffer

4 ml tetrahydrofuran

0.30 ml diethyiethcl'

.} Sediment

(organic phase)

t addition of:

t Centrifugation

I Supernatant

(aqueous phase)

0.1 volume of distilled water ,),

Centrifugation

I t t

Sediment Supernatant

(aqueous phase) I +

Combined supernatants

t Dialysis against distilled water

Gangliosides on the dialyzed aqueous phase were evaluated by colorimetric procedures for sialic acid (WARREN 1959) and separation by TLC (see below).

c) Determination of proteins

Protein concentration was tested according to LOWRY'S procedure (1951) on the defatted

sediment. For this procedure the sediment was dissolved in 0.1 N NaOH and diluted with distilled

water. Bovine serum albumine (Si gma) was used as the standard.

d) TLC separation

Phospho- and sulpholipids: the organic phase was used for separation of phospholipids and

sulpholipids. Chloroform, methanol, water (70: 30: 5 by vol.) was used a8 the solvent. Ganglio­

sides: aqueous phase (see page 14) was used for the separation of gangliosides. Chloroform,

m ethanol, water (60: 35: 8 by voL) was used as the solvent. Spots were detected by exposure to iodine vapour or spraying with anisaldehyde reagent. Gangli08ides were also detected by spraying with Ehrlich reagent (p-dimethylaminobenzaldehyde)_

3. Histochemical reactions

Enzymatic tests were performed in order to reveal histochemical evidence for enzyme activity known to be involved in processes associated with intense osmotic work in cells exposed to hyper­

tonic medium.

Biochemical and histochemical features 15

Monolayer cells were grown on coverslips in PETRI plastic dishes. Both EUE cells grown on

hypertonic medium (0.356 M) and on isotonic medium (0.137 M) were tested.

The culture medium was discarded and the cells on the bottom were washed in 0.356 M NaCI

and in 0.137 M NaCl respectively. The monolayer cells were tested for the following enzymatic

activities.

a) Oxoreductases

Lactate and glucose.6.P.dehydrogenases (10- 2 M substrate; 10-3 M cohenzyme and 0.25 % Nitro BT in 0.2 M phosphate buffer pH = 7.4 (PEARSE 1972).

NADH·dependent diaphorases (10-2 M substrate, 0.25 % Nitro BT). Tetrahydrofolate reductase (10- 2 M substrate dissolved in 0.1 % 2.mercaptoethanol, 10-1 M

NADP, M/15 phosphate buffer to get pH = 7.4, 0.3 mM Nitro BT, 0.2 mM phenazine methosul· phate, 5 X 10-3 M MgCl2 and 10-3 M NaN 3; 5 mM Amethopterine was added as control (GER.

ZELI and DE PrCEls 1972).

b) Hydrolases

Acid phosphatase according to Bl:RSTONE'S procedure (1961) (Naphtol phosphate in acetate

buffer 0.1 M, pH = 5.2 and Fast red violet LB as coupling reagent). Alkaline phosphatase was assayed either by BURSTONE'S procedure (Naphtol phosphate in

0.05 M, pH = 8.3 Tris maleate buffer) or by GOMORY'S procedure [(1914); (Na.glycerophosphate

3 % in Veronal sodium buffer 2 % with ammonium sulphide as reducing reagent)].

Mg++ dependent ATPase at pH = 7.2 (3 X 10-3 M ATP in Tris buffer, II X 10-3 M Mg++,

3 X 10-3 Pb as trapping agent). Activation was performed by adding NaCI and Kel and inhibition

control by adding Ouabaine [(3 X 10-5 M); (MCCLURKIN 1964)]. The monolayer cells were also tested for:

Polysaccharides: PAS reaction, Alcian blue pH = 3.5 reaction.

Proteins: Bromophenol blue rRaction. Polar lipid8: Sudan black B.

Results

The growth rate of cells maintained in the hypertonic medium was evaluated on the basis of the number of cells per colony developed from single cells after 6 days of culture. Cells cultured in 0.274 M NaCl (treated in hypertonic medium) show a slower growth rate (13.50 ± 0.60 cells/colony) than those in the controls grown in 0.137 M NaCl (48.60 ± 1.64 cells/colony). Plating efficiency did not show any appreciable change as between the treated and control cells (0.21 ± 0.01 for treated cells, 0.194

± 0.004 for the controls).

A. Biochemical analysis

The results of polar lipid evaluation are reported in Tables 1, 2, 3. The phospholipid concentration in ~amples of cells grown in 0.274 M NaCI does not

differ appreciably from that in the controls, although a slight increase in phospho. lipids occurs in the former group after 40 to 70 days culture (Table 1).

Differences between treated and control cells are appreciable as far as sulpholipids and gangliosides are concerned (Tables 2, 3).

16 L. BOLOGNANI et al.

Table 1. Determination of phospholipids

Days fig P Lipid boundjmg protein

Hypertonic Isotonic

mcdium medium

0 ... 7 7.9 12.1

8.2

8 ... 28 8.9 12.0

6.2

29 ... 42 9.8 8.9

43 ... 56 15.7 8.5

57 ... 70 8.3 13.0

Table 2. Determination of sulfolipids

Days fig sulfolipidjmg protein

Hypertonic Isotonic

medium medium

0 ... 7 22.4 20.7

24.9 8 ... 28 33.4 25.3

69.1 29 ... 42 77.0 19.2

93.7 43 ... 56 86.0 28.9

69.2

57 ... 70 60.4 32.0

55.0

Table 3. Determination of gangliosides

Days nmoles of sialic acidjmg protein1)

60 75 80

Hypertonic

medium

1.7 2.6

13.0

Isotonic medium

0.5

0.5

1) Protein concentration ranges from 1.10 to 1.30 fig X 1O-4 jcells in controls and from 0.9 to 1.7 fig X 1O-4 jcells in treated samples.

Biochemi<:al and histochemi cal features 17

F

o Ev

c T 5 Fig. 1. TLC separation of gangliosides in aqueous phase f!'Om lipid extracts of (T) treated cells

(0.274 M NaCl) and (0) coni l'Ob (0.137 !vI NaCI) .

. . o 5

o

.: ;", . .

... ...... .

Fig . 2. Scanning ofTLC plates.

::: :

.. ...

... .

"5 io

o control

........•... : ... ......... .

• treated

." ..

SuJpholipid content in cells grown on hypertonic medium increases with time. A fourfold increase in sulpholipids is evident in cells grown on 0.274 M NaCl for 2 months, as compared with the controls.

An even more marked increase in gangliosides was found in cells grown on hyper­tonic medium.

Moreover the ganglioside pattern appears to be qualitatively modified as can be judged by inspecting and comparing TLC plates prepared from aqueous phases of lipid extract" (Fig. I). ~ Acta hi storh elli. Bd. 62

18 L. BOLOGNANI et al.

o AQUEOUS PHASE

Fig. 3. Preparative TLC of gangliosides from aqueous phase of EUE cells.

S

GM3 GM2 GM1

G D1a G D1b Gr GQ

The scanning of chromatogram slides by a Joyce scanner (ratio 1: 5, slit 18: 381 filter) clearly demonstrates quantitative and qualitative differences in the ganglio­sides (Fig. 2).

The most evident fraction in both groups has been tentatively identified on the basis of Rf value as being polysialoganglioside GT in preparation TLC (Fig. 3).

An unidentified spot appears in extracts of cells grown on hypertonic medium; its Rf is intermediate GMI and GD l .

B. Histochemical reactions

In the histochemical tests for polysaccharides, proteins and lipids there are no differences as between EVE cells in hypertonic medium and those in the isotonic

ilioehernit'al and histoehernieal features

Fig. 4. N ADH dependent diaphorase ( < 1250).

A.) Treated cells; B) Contl'ols.

19

medium. Lactate, gluco:-;e-o-P-dehydrogena:-;e and NADH diaphorase appear to be

strongly enhanced in cell:-; grown on 0.356 111 NaCl [(reacting granula are well defined in the cytoplasm); (Fig::;. 4, 5) j.

In the monola.ver cells submitted to N ADH diaphorase less coloured cells appear in both groups. Tetrahydrofolate reductase in the normal cells appears as fine granula and round-shaped bodit::<. Tn the cells grown on hypertonic medium the enzyme reaction i" more intell"p, in all the cytoplasm but the defined granula Of spherical shaped bodie:< art: not appreciablt: (Fig. 6).

:!.*

20

Fig. 5. G-6-P dehydrogenase (x 1250). A) Treated cells; B) Controls.

L. BOLOGNANI et al.

Higher acid phosphatase activity was found in the cells grown on isotonic medium (0.137 M NaCl) than those grown on hypertonic medium. In both groups some cells appear negative. No alkaline phosphatase was demonstrable in about 75 % of the cells in both groups and positivity was less pronounced in the cells grown on hyper­tonic medium.

ATPase activity is hardly demonstrable in hypertonic cells (Fig. 7). This finding is surprisingly different from that observed in glands exposed to stronger osmotic conditions (ATPase is stronger in these glands than in resting glands). Findings on hydrolytic enzymes are the opposite of those in oxoreductases since hydrolytic enzymes

Biochemical and hi stochemical features

Fig. O. Tetrahydrofolate retiuct,ase ( / ·!UO).

A) Treated cells; B) Control s.

21

are stronger in the cells growing in isotonic medium than in those growing in the hypertonic medium. Conversely, the oxoreductases are stronger in the hypertonic cells.

Discussion

Our bioche mical ,·",mlts a,'e con sis tent with those of previous experiments carried out on ver ­

tebrate organs involved in regula ting ~alin t> homeostasis (KARLSSON et al. 1968; BOLOGNANI 1970;

BOLOGNANI et al. 1976; GERZELI et. al. 1976). On the contrary, the histochemical result,s are not

"ompletely in agreement with tho~e pre\'iously reported for these organs. Our efforts were directed at est a blishing whether some polar lipid s, e,g., sulpholipids, increase

in eell cultures exposed to hypertoni e ~aline solution. in a simila,' manner to what occurs in vertebrate glands during adaptation to hY]ll'I'tonic l'nvironmental conditions.

22 L. BOLOGNANI ot a!.

. •

Fig. 7. Membrane ATPase [(Na +, K + dependent); ( X 460)].

A) Treated cells; B) Controls.

Furthermore, cytological and cytoenzymatic tests were carried on cell cultures in hypertonic

llledium to demonstrate any analogie or differences vis-a-vis vertebrate glands exposed to strong oSlllotic work. Cytological observations of structures involved in osmotic work reveal intense protein and phospholipid staining, the presen ce of acidic GAG on the cell boundaries, a high level of oxoreductases and the presen ce of Mg + + ATPase.

Our results indicate that sulpholipids increase in the cells treated with 0.275 M NaCl; the

sulpholipid concentration reaches a maximum (400 % over the controls) after 50 to 60 days cul­ture. An incr ease in ganglioside concentration is also demonstrable in about the same period of

time, but higher ganglioside concentration was observed in 80·day cell cultures.

Biochemical and histochemical features 23

Spots with Rf~ comparable to Trisialoganglioside, i.e. a polysialoganglioside, are present

III both groups of this human cell line. Scanning of TLC plates indicates that the ganglioside

pattern in the EUE·treated cells is also qualitatively different to that in the controls.

These changes in glycolipids may be regarded either as a defence mechanism in themselves

or as a sign of a more coordinated cell defence mechanism as a whole. According to ABRAHAMSSON

(1972), sulphatides are the only polar lipids that undergo conformational changes in vitro within

physiological limits of temperature and hydration. Hydration conditions are certainly affected

by an increase in tl18 tonicity of the medium (CERBON 1972), particularly if hypertonicity is ob­

tained by an increase in ion concentration, as was the case in our experimental conditions (Bo­

LOGNANI et al. 1976).

The increase in gangliosidcs probably reflects similar conformational changes. We have no

comparable data available for vertebrate glands. No appreciable changes in phospholipid con­

centration were observed in either group. These results are consistent with those reported for

glands adapted to eonsidcrabk osmotic work (GERZELI et al. 1971, 1973; KARLSSON et al. 1974;

BOLOGNANI et al. 1976).

Cytoenzymatic tcsb' on EUE cells treated with hypertonic medium revealed a high level of

oxoreductase activity as observed in the glands. This response is likely to be independent from the

general response of til<' organism as a whole, since single cells are capable of showing marked in·

creases in oxoreductase activity.

The biological meaning of this increment is still open to discussion. The higher oxoreductase

activity in cells exposed to hypertonic medium and in the glands is generally considered to be re­

lated to encrgy requiremcnts for osmotic work.

In addition to strudural changes, mechanisms for "energy saving" are also likely to support

the survival of EUEs adapted to hypertonic saline solutions. This may explain the slower growth

rate of EUEs adapted to hypertonic medium than the controls.

The lower ATPase activity in the lst group may also contribute to saving ATP. The lower

ATPase activity we have found in cells exposed to hypertonic medium is apparently in contrast

with findings in vertpbrate organs; in fact, Na + and K + activated ATPase is higher in the organs

of vertebrates adapted to hypertonic saline conditions and in specimens living in the sea than those living in lakf's.

The low ATPase adi\"ity may be due not so much to the lack of enzyme, or its destruction, but to the unavailability of active sites because of changes in molecular conformation. We remain

cautious in our interpretation of ATPase results, in view of recent evidence that questions the

validity of the Lead methods used to test N a + and K + activated ATPase by histochemical pro­cedures.

The decrease in hydrolase activity seems to be a more generalized condition in EUE growing

on hypertonic medium. since acid and alkaline phosphatase also appear to decrease.

It should be mentioned that two cell populations are present in both groups of this EUE

transformed line as prC'\"iously described by DE CARLI et al. (1964): phosphatase+ and phosphatase­

cC'lls are present in ECE grown on both hypertonic and on isotonic medium. The decrease in the

intensity of acid phosphatase activity in EUE cells exposed to hypertonic medium may possible

be explained if one rC'ea\l" that, sulphuric e,;ter,; (such as sulpholipids) are "cross-inhibitors" of

acid phosphat.ases (BOLOGXANI et al. 1973, 1976). The possibility of similar inhibition in alkaline

phosphatase has not ypt been explored.

Tests for several different hydro lases are in progress on both groups in order to ascertain

whether the decrease in hydrolases activity is a general phenomenon in cells exposed to hypertonic

conditions, possibly to a\"oid water consumption in splitting substrat.es (in as much as water is required for osmotic proeesses).

It is difficult to demonstrate whether any relation exists between the increase in oxoreduc­

tases and the decrease in ATPases. Certainly, EUE cfllls surviving in 0.274 M NaCI derive energy to perform aetin' t,ransport through their membranes from oxoreductive reactions.

24 L. BOLOGNANI et al.

The question arises as to whether this energy derives directly from oxoreductive processes

(without transphosphorylating reactions or despite very low transphosphorilating reactions) in EUE cells surviving in 0.274 M NaCI.

Oxoreductive processes directly supplying energy occur in bacterial cultures. If such processes

are also demonstrated in our case considerable support for the chemioosmotic hypothesis will be provided by cell cultures.

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Address: Prof. dott. ANNA MARIA BOLOGXANI FANTIN, Cattedra di Istologia ed Embriologia,

Via Berengario 14, I - 41 100 Modena.