/. Embryol. exp. Morph. Vol. 30, 2, pp. 511-518, 1973
511
Printed in Great Britain
Measurements of cell adhesion
II. Quantitative study of the effect of divalent ions
on cell adhesion
By JANET E. HORNBY 1
From the Department of Zoology, University of Reading
SUMMARY
The forces of interaction found for 5-day chick limb-bud cells in media containing different
divalent ions (magnesium, calcium, strontium or barium) show that the cells are most
adhesive in the presence of magnesium ions, then calcium and strontium, then barium. When
magnesium and calcium ions are present together at similar concentrations the calcium ions
modify the action of the magnesium ions. The importance of magnesium ions in cell adhesion
is discussed.
INTRODUCTION
The role of divalent ions in cell reaggregation has been investigated in order
to further test the assumption that suspensions of dissociated embryonic cells
behave as colloid systems. The Derjaguin & Landau (1941) and Verwey &
Overbeek (1948) theories of colloid stability are based on an appreciation of the
repulsive forces due to like charges on the particles and the London van der
Waals attractive forces. The thickness of the double layer of counter ions and
therefore the shape of the repulsive potential curve depends both on the concentration and the valency of the electrolyte in the continuous phase. The ratio
of the concentrations of monovalent: divalent itrivalent ions required to
flocculate a given sol has been found by calculation (Verwey & Overbeek,
1948) and experiment (Schulze, 1882, 1883; Hardy, 1910; Lychnikov &
Martynov, 1965) to be of the order of 10 3 :10:1. This phenomenon may account
for the importance of extracellular divalent ions in the maintenance of
adhesion between embryonic cells (Herbst, 1900; Rous & Jones, 1916; Anderson, 1953; Zwilling, 1954).
The apparent force of adhesion between 5-day chick limb-bud cells was
found for a suspension of cells in a physiological saline (Hanks' +199, containing 1 -3 HIM calcium ions and 0-9 mM magnesium ions), a saline free from divalent
ions (CMF Hanks') and in solutions in which the calcium and magnesium salts were
replaced with 2-2 mM of one divalent salt (Ca2+ or Mg 2+ or Ba2+ or Sr2+ Hanks'
1
Author's address: Department of Zoology, University of Reading, Whiteknights, Reading
RG6 2AJ, U.K.
512
J. E. HORNBY
saline) to see if aggregation depended on calcium and magnesium ions, or if
these ions may be replaced by any divalent ion.
METHODS
1. Biological methods
Cell suspensions were prepared from the limb-buds of 5-day chick embryos
(White Leghorn) as described in the previous paper (Hornby, 1973), in CMF to
a population density of approximately 8-4 x 106 cells/cm3. At the beginning of
the experiment the cell suspension was added to the test solution in a 10 cm3
siliconed, conical flask (1 cm3 suspension to 5 cm3 of solution) to give an
approximate density of 1-4 x 106 cells/cm3. The cell density was determined
using a Fuchs-Rosenthal haemocytometer.
The suspensions, in the 10 cm3 flasks, were shaken backwards and forwards
at a fixed rate (80 rev/min) in a Gallenkamp shaking reaction incubator 1-H-35O,
maintained at 37 °C. Samples were taken from the flasks at regular intervals
using wide bore Pasteur pipettes, and the single cell density was counted in
a haemocytometer.
At the end of the experiment the aggregates were resuspended in CMF and
then added to a medium containing 5 % horse serum, 5 % embryo extract,
45 % 199 and 45 % Hanks', and plated out in Falcon Petri dishes. The dishes
were examined next day for cells that had settled and spread. Settling and
spreading was taken as an indication of a living cell.
The experimental suspension media were tested for the presence of calcium
ions on a flame spectrophotometer (Unicam S.P. 90).
In a further series of experiments several cell suspensions were prepared in
one of the experimental suspension media. The cell suspensions were shaken
and then, after different time intervals, the suspensions were centrifuged, the
cells were resuspended and plated out as above, and the supernatant was saved
and analysed for calcium. Unused suspension media were also analysed for
calcium.
2. Computation
The experimental data were grouped according to the typs of suspension
media used. Considering the application of the modified Smoluchowski relationship (Hornby, 1973) to the data, the gradients of the lines obtained within each
group should be parallel since the energy supplied was constant throughout
the experiments. The best individual and parallel lines between l/v(Wx0 vi) a r | d
t were fitted to the data using a programme for comparison of regressions. The
calculated and experimental intercepts, and the variance about the parallel and
individual lines, were compared. The apparent net Hamaker coefficients of
attraction, and the potential and force of interaction for cells in the different
media, were calculated from the gradients of the parallel lines assuming a
distance between the two surfaces of 2 nm (Hornby, 1973).
Measurements of cell adhesion. II
513
10
100
200
30 r C
30 - D
20
20 -
10
10
>
i
0
100
200
300
0
30 r E
30 r F
20
20
l
i
100
200
300
100
200
300
10
100
200
300
0
/ (min)
Fig. 1. Examples of the relationship between l/V(Nooo vO and /for 5-day chick
limb-bud cells aggregating in (A) Hanks'+199; (B) CMF Hanks'; (C) magnesium
Hanks'; (D) calcium Hanks'; (E) strontium Hanks'; (F) barium Hanks'.
,
Individual line; - - , parallel line.
RESULTS
V
I) plotted against / gave a good straight-line relationship, as illustrated in Fig. 1. The experimental intercept fell within the 5% confidence
limits of the calculated intercept in 27 out of 29 cases. This was used as an
informal test for linearity.
Comparison of the variance about the individual and parallel lines showed
514
J. E. HORNBY
Suspension medium:
Hanks'+199
CMF Hanks'
Magnesium Hanks'
Calcium Hanks'
Strontium Hanks'
Barium Hanks'
0001
0002
0003 0004 0005
a Y (sec" ')
0006
Fig. 2. Values o f a y , with 95 % confidence limits, for 5-day embryonic
chick limb-bud cells in different suspension media.
Table 1. Collision efficiencies, energies and forces of interaction of
5-day chick limb-bud in presence of different divalent ions
Suspension
medium
n
H+199
CMF
Ca 2+ Hanks
Mg2+ Hanks
Ba 2+ Hanks
Sr2+ Hanks
6
6
6
6
6
5
Collision
efficiency
coefficient
(in J)
VA
(J/m2)
9-62
919
5-88
10-35
4-85
5-93
1-3 xlO- 24
1-lxlO- 24
2-lxlO- 2 5
l-8xlO- 24
l-2xlO" 25
2-2 xlO- 25
8-9 x 1G7-3 x 10 9
l-4x 10~9
l-2x io- 8
7-7 x 1Q-10
1-5 x io- 9
Hamaker
VA
(inJ)
9
4x
3x
6x
5x
3x
6x
10 19
10 -19
10 20
10 19
10- 2 0
10 20
FA
(N/m 2 )
8-9 x 10°
7-3 x 10°
1-4 x 10°
l-2x 10l
7-7 x 10 x
l-5x 10°
FA
(in N)
4 x 10
3x 10
6 x 10
10
10
11
5x 10
10
3x 10
11
6x 10
11
Shear rate 1 -74 sec"1.
no significant difference in four out of six cases, and the remaining two showed
only 5 % significance. It can be seen from the values of a Y, and their 95 %
confidence limits (Fig. 2) that the six sets of data fall into two groups: H + 199,
CMF and Mg 2+ ; and Ca2+, Ba2+ and Sr2+. The apparent Hamaker coefficient and
the energy and force of interaction between the cells in the different experimental
suspension media are given in Table 1.
The cells that had been shaken in H +199 settled and spread. The survival of
the cells that had been shaken in abnormal media, as tested by this method, was
variable but in the subsequent experiment it was found that the cells would
survive to settle and spread after 100 min in abnormal media.
When the experimental suspension media were analysed for calcium ions in
a spectrophotometer the supposedly calcium-free solutions CMF, Mg2 +, Ba 2f
and Sr2+ Hanks' were found to contain approximately 0-01 HIM calcium ions.
H + 199 contained approximately 1-2 mM calcium. The calcium in the calcium-
Measurements of cell adhesion. II
515
free solutions had apparently not leaked from the cells since very small amounts
of calcium were found in the unused suspension media, and the amount did
not increase after cells had been in the suspension media for given time intervals.
DISCUSSION
The values found for the adhesive energy and forces of interaction between
the cells suggest that the cells are more attractive when suspended in H + 199,
CMF and Mg 2+ Hanks' than when suspended in Ca2+, Ba2+ or Sr2+ Hanks'.
The forces of interaction between the cells are as great in solutions containing
very small amounts of divalent ions (001 ITIM) or a high concentration of
magnesium ions (2-2 ITIM), as they are in solutions containing the normal
extracellular balance of calcium and magnesium ions. In the presence of a low
concentration of magnesium and a high concentration of calcium, barium or
strontium ions the forces of interaction are reduced. Therefore the role of
divalent ions in cell adhesion does not appear to be a non-specific effect of
divalent ions on the diffuse double layer around the cells and hence on the
repulsive potential.
Stern (1924) modified the Gouy Chapman theory of the diffuse double layer
to take account of ionic size and also proposed the specific adsorption of a
layer of counter ions near the surface of the particles. The adsorption of
divalent ions at the Stern layer could explain the effects of the different divalent
ions in cell adhesion.
Magnesium, calcium, strontium and barium show selectivity sequences at
biological membranes which agree with seven theoretical sequences based on
the difference between the free energy of interaction between the cation and
the surface, and the free energy of interaction between the cation and water
(Diamond & Wright, 1969). Selective adsorption of divalent ions at the Stern
layer could result in a quantitative sequence of attractiveness in the presence of
different divalent ions. Disregarding the overlapping confidence limits of the
Hamaker coefficients, the values for the force of interaction (Fj) between 5-day
chick limb-bud cells in magnesium, calcium, barium and strontium Hanks'
follow the sequence:
Mg2+ (5 x 10-10 N) > Ca2+ (6 x KH 1 N) = Sr2+ (6 x 10"11 N)
> Ba2+ (3 x 10"11 N),
which is close to one of the seven possible sequences for divalent ions considered
by Diamond & Wright (1969): Mg 2+ > Ca 2+ > Sr2+ > Ba2+. This suggests
that the force of interaction between the cells could be controlled by selective
adsorption of divalent ions at the Stern layer.
The reduction in the force of interaction found between cells in Hanks'+199
(4x 10 1 0 N), which contains calcium and magnesium ions in the ratio of 4:3,
516
J. E. HORNBY
compared with that found in magnesium Hanks' (5 x 10~10 N), may be due to
competition for adsorption by calcium ions.
Selective adsorption at the Stern layer does not explain the high values found
for the force of interaction between cells in CMF (3 x 10~10 N).
An explanation of the effects of divalent ions on the adhesion of cells in terms
of selective adsorption at the Stern layer implies a reduction in the net negative
surface charge density in the presence of these ions at physiological concentrations. Lipson, Dodelson & Hays (1965) working on toad bladder epithelial
cells showed that all the multivalent ions they tested (calcium, cadmium, cobalt,
magnesium and barium) reduced the negative surface charge of the cells. Using
the restoration of ohmic resistance to the cells as their criterion for assessing
adhesion, they also considered the effect of various multivalent ions on adhesion;
calcium and strontium were able to restore resistance, magnesium and barium
were not. Lipman et al. (1966) found that concentrations of calcium or magnesium
as low as 1 niM and 5 mM would considerably reduce the surface charge of toad
bladder epithelial cells. Collins (19666) showed that calcium ions are necessary
for the reaggregation of 7-day chick neural retinal cells, and that increasing
extra cellular calcium concentration progressively reduced the net surface charge
density so that at physiological concentrations calcium had a considerable effect
in reducing the net surface charge density of these cells. Gingell and Garrod
(1969) found that when cells at the pre-aggregate stage of the slime mould
Dictyostelium discoideum were incubated in either phosphate buffer containing
5 mM MgCl2 or the same buffer plus 10~3M EDTA the surface charge of the cells
increased slightly after incubation with EDTA and the cells did not adhere. It
would appear that reduction in cell surface charge does not always result in
adhesion but as emphasized by Gershman (1970) different methods of measuring
adhesion often measure different facets of adhesion.
The correlation between surface charge reduction and relative strengths of
adhesion is also equivocal. Collins (1966a) compared the reduction of surface
charge by given calcium concentrations for 5-day liver and heart and 8-day
epidermis of chick embryos with Steinberg's heirarchy of adhesiveness for these
cell types (liver < heart < epidermis). The surface charge of the heart cells was
decreased twice as much as that of the liver cells suggesting a correlation between
surface charge reduction and strength of adhesion. The surface charge reduction
for epidermal cells was no greater than for liver cells, yet epidermis is more
adhesive than liver or heart. Steinberg's measure of adhesion assumes, however,
that relative strengths of adhesion are involved in selective adhesion of cell types.
The reduction of surface charge may be involved in aligning the cell membranes
but selective adhesion may require the establishment of low resistance junctions
and communication between cells (Hornby, 1973).
Other workers have also shown that magnesium is apparently more effective
in cell adhesion than calcium. Armstrong (1966) observed both the aggregation
and settling out behaviour of 4-day chick limb-bud cells in very simple solutions
Measurements of cell adhesion. II
517
containing either only 0-145 M sodium chloride, or a 0-145 M solution containing
calcium or magnesium ions and sodium chloride. Under these conditions the
cells are shown to aggregate faster in both calcium- and magnesium-containing
solutions but the magnesium solution was apparently more effective than the
calcium solution. When the cells were plated out in a magnesium solution the
cells quickly spread and adhered to glass, whilst the cells that were plated out
in a calcium (or barium or strontium) containing solution remained rounded up.
Similarly Rous & Jones (1916) observed that if cells growing in a plasma clot
were washed in Locke's solution, which contains 2-2 mM calcium but no
magnesium ions (Paul, 1965), the individual cells contracted into spheres
within the meshwork of the plasma clot. Jones (1966) considers that when
unbalanced by each other calcium ions cause contraction, and magnesium ions
relaxation of the actomyosin-like substances found at cell peripheries, and that
this might result in an increased cell-surface charge density in calcium solutions.
Calcium is, however, known to be important in cell adhesion; removal of
calcium from the extracellular environment of many tissues results in their disaggregation. Daday & Creaser (1970) extracted a protein from normal retinal
cells by incubating the cells in 0-05 M EDTA. This protein was found to be
necessary and specific for the re-aggregation of normal retinal cells. Hays et al.
(1965) showed that the removal of calcium from media surrounding intact toad
bladder tissue resulted in the disappearance of the extensive junctional complexes and desmosomes leaving individual cells rather than an intact tissue.
Calcium and magnesium ions appear to be able to replace one another in the
establishment of low resistance junctions (Loewenstein, Nakas & Socolar, 1967).
It seems that the importance of divalent ions in cell adhesion cannot be
entirely explained in terms of the theory of lyophobic sols, but during the initial
phase of re-adhesion magnesium ions seem to be very important and calcium
ions appear to modify the strength of the adhesion between the cells.
I am most grateful to Professor A.S.G.Curtis for supervising this work which was carried
out during the tenure of an S.R.C. Research Studentship in the Department of Zoology,
University College, London, and completed in the Department of Zoology, University of
Reading. I am grateful to Professor M. Abercrombie and Professor A. Graham for the
facilities provided. I should also like to thank Mr D. Arnold for technical assistance at
University College, London, and Mr R. Stern of Department of Applied Statistics, University of Reading for valuable assistance with the statistical analysis.
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(Received 14 July 1972, revised 14 May 1973)