lipoproteins bound to the cell membrane or 37°C cell association, in which lipoproteins are associated with
both cell membrane and intracellular compartments, in the absence or presence of exogenous LPL. Commer-
cially purchased RPMI-1640 medium normal RPMI- 1640 medium contains 0.42 mM Ca
2 +
and 0.4 mM Mg
2 +
. CaCl
2
· 2H
2
O and MgCl
2
· 6H
2
O were added to this medium to achieve the desired final concentrations
of Ca
2 +
and Mg
2 +
. At the concentrations of Ca
2 +
and Mg
2 +
used, there was no precipitation in the medium as checked by absorption at 450 nm. In some experi-
ments, 1.34 mM EDTA was added to the medium to chelate Ca
2 +
and Mg
2 +
in the medium. After washing, the cells were dissolved with Triton X-100 0.05.
Fluorescence in the cell lysate was measured in tripli- cates in enhancement solution with a VICTOR™ Mul-
tilabel Fluorescence Counter Wallac Oy. Cell protein content was measured in duplicate with the method of
Lowry et al. [11] using BSA as standard. Specific cellu- lar binding and association were calculated by subtract-
ing the non-specific binding or association in the presence of an excess of unlabeled lipoproteins from
those in the absence of unlabeled lipoproteins.
Binding of biotin-labeled LPL to MPM was carried out in RPMI-1640 medium containing different concen-
trations of Ca
2 +
or Mg
2 +
, for 2 h at 37°C. As control, biotin-labeled LPL was omitted from the medium. Af-
ter washing with PBS, Eu
3 +
-labeled streptavidin in RPMI-1640 medium was added to the cells and incu-
bated for 30 min at 37°C. Cells were then washed and the fluorescence was measured as in cell binding and
association studies. Specific binding was calculated by subtracting the fluorescence counts in the absence of
biotin-labeled LPL from those in the presence of biotin- labeled LPL.
2
.
7
. Measurement of cellular cholesterol MPM were cultivated in 24-well plates Costar in
RPMI-1640 medium containing 10 FCS. Twenty four hours before the experiment, RPMI-1640 medium sup-
plemented with 10 human LPDS instead of FCS was added to the cells. At the start of the experiment, the
cells were washed three times with RPMI-1640 medium. RPMI-1640 medium containing 1 BSA, 100 mgml of
native and oxidized LDL, various concentrations of Ca
2 +
or Mg
2 +
, in the presence or absence of 10 mgml LPL, was then added to the cells and incubated for 24
h in a humidified incubator with 5 CO
2
at 37°C. Control incubations were performed in RPMI-1640
medium containing 1 BSA without other additions. At the end of the incubation, the cells were washed
three times with PBS containing 0.1 BSA and two times with PBS. Cellular total and free cholesterol
content were measured [17].
2
.
8
. Binding of Eu
3 +
-labeled nati6e and oxidized LDL to LPL
An aliquot of 1.5 mg of LPL from bovine milk in 100 m
l PBS was coated to each well of the microtitration plates Nunc at 4°C for 18 h. After three washes with
PBS, each well was blocked with 200 ml PBS containing 3 BSA for 1 h at room temperature. The plates were
washed three times with PBS, and then RPMI-1640 medium or Tris – HCl buffer containing 50 mM NaCl,
1 BSA, Eu
3 +
-labeled native and oxidized LDL, and different concentrations of Ca
2 +
or Mg
2 +
was added to each well. After incubation for 1 h at 37°C, the wells
were washed with PBS, and the fluorescence of bound Eu
3 +
was measured in the presence of enhancement solution 200 mlwell. To measure the non-specific
binding, bovine serum album instead of bovine milk LPL was coated to the plates, and the binding was
measured the same way. The non-specific binding of Eu
3 +
-labeled lipoproteins to BSA as compared to LPL was below 5, and was subtracted from the amount of
binding to LPL.
3. Results
3
.
1
. LPL-mediated binding and cell association of Eu
3 +
-labeled nati6e and oxidized LDL to MPM in the presence of different concentrations of Ca
2 +
and Mg
2 +
The effect of LPL and increased concentrations of Ca
2 +
in the medium on the binding of native and differently oxidized LDL to MPM was studied Table
1. In the original RPMI-1640 medium containing 0.4 mM Ca
2 +
, the binding of Eu
3 +
-LDL to MPM in- creased with the degree of oxidative modification. The
percentage of the enhanced binding caused by addition of LPL or 1.4 mM Ca
2 +
to this medium decreased with the degree of oxidative modification of Eu
3 +
-labeled LDL. The combination of LPL supplement and the
addition of 1.4 mM Ca
2 +
in RPMI-1640 medium led to a binding higher than the sum of the separate effects
of LPL and increased Ca
2 +
concentration for both native and oxidized LDL, i.e. LPL and Ca
2 +
synergis- tically increased the binding of native and oxidized
LDL to MPM Table 1. It is noteworthy that LPL and the increased Ca
2 +
concentrations, although each sepa- rately had little effect on the binding of extensively
oxidized LDL to MPM, when combined increased the binding of this oxidized LDL by 78.3. When the
increased binding due to the separate addition of LPL and Ca
2 +
to the medium was subtracted from the binding achieved by the simultaneous addition of LPL
and 1.4 mM Ca
2 +
to the medium, the synergistic effects were found to be the highest for 1 and 4 h
Table 1 Effect of LPL and Ca
2+
concentrations on the binding of native and differently oxidized LDL to MPM
a
RPMI+LPL RPMI+1.4 mM Ca
2+
RPMI+LPL+1.4 mM Ca
2+
RPMI control Synergistic effect
8.13 9 0.2 546.5 6.38 9 0.36 407.6
15.85 9 1.22 1159.5 LDL
2.58 205.4 1.26 9 0.20
11.11 9 0.46 117.9 10.83 9 2.33 112.3
5.10 9 0.34 35.60 9 0.15 597.9
oxLDL 1 h 18.73 367.7
17.36 9 0.39 102.2 12.62 9 1.30 47.0
oxLDL 4 h 57.33 9 2.11 568.0
8.58 9 0.31 35.94 418.8
55.73 9 16.61 30.8 57.03 9 0.68 33.9
42.59 9 0.32 141.82 9 2.70 233.0
oxLDL 8 h 71.66 168.3
433.02 9 9.51 1.8 433.10 9 2.85 1.8
758.34 9 17.66 78.3 317.57 74.7
oxLDL 24 h 425.35 9 10.54
a
Binding of Eu
3+
-labeled native and differently oxidized LDL to MPM was measured in the original RPMI-1640 medium containing 0.4 mM Ca
2+
set as control, or in this medium with the addition of LPL 10 mgml, or with the addition of 1.4 mM Ca
2+
, or with the addition of both LPL 10 mgml and 1.4 mM Ca
2+
. After 4 h of incubation at 4°C and washing the cells, the binding was measured ng LDL proteinmg cell protein. Synergistic effects were calculated by subtracting the separately increased binding in the presence of LPL and the binding in the presence
of additional 1.4 mM Ca
2+
concentration from that in the presence of both LPL and additional 1.4 mM Ca
2+
. Values represent mean 9 SD of three experiments. Values in parentheses represent the percent increase compared with the controls, or the percent synergistic effect the last
column of LPL and the increased Ca
2+
concentration. PB0.01 as compared with controls.
oxidized LDL. In contrast, the addition of 1.4 mM Mg
2 +
in the medium neither influenced the binding of native and differently oxidized LDL to MPM, nor
affected the LPL-mediated binding of native and oxi- dized LDL to MPM not shown. The results obtained
from cell association studies were similar not shown.
3
.
2
. Binding of biotin-labeled LPL to MPM in the presence of different concentrations of Ca
2 +
and Mg
2 +
In order to evaluate the influence of Ca
2 +
and Mg
2 +
on the direct binding of LPL to MPM, biotin-labeled LPL was used. As shown in Fig. 1, there was no
significant difference in the binding of biotin-labeled LPL to MPM, when either EDTA was used to chelate
divalent cations in RPMI-1640 medium, or an addi- tional 1.4 mM Ca
2 +
was added to this medium P \ 0.2. Moreover, the addition of 1.4 mM Mg
2 +
to the medium decreased the binding of biotin-LPL to MPM
by 43 P B 0.05.
3
.
3
. Effect of LPL on the accumulation in MPM of esterified cholesterol deri6ed from nati6e and oxidized
LDL in the presence of different concentrations of Ca
2 +
The effect of LPL on the accumulation of esterified cholesterol in MPM in the presence of different concen-
trations of Ca
2 +
was studied. Free and total cholesterol in MPM were measured after incubating the cells for 24
hours in RPMI-1640 medium containing 100 mgml of native and differently oxidized LDL and different con-
centrations of Ca
2 +
in the absence or presence of LPL. Esterified cholesterol was calculated by subtracting the
free cholesterol from total cholesterol. As shown in Table 2, the addition of LPL or 1.4 mM calcium to
RPMI-1640 medium both increased the accumulation of esterified cholesterol derived from native LDL and
mildly or moderately oxidized LDL in MPM, while both exerted only small effects in the case of extensively
oxidized LDL. The combination of LPL and the in- creased calcium concentration in the medium led to a
cholesterol deposition that was higher than the sum of the separate effects of LPL supplementation and in-
creased Ca
2 +
concentration. Thus, LPL and Ca
2 +
Fig. 1. Effect of the Ca
2 +
and Mg
2 +
concentrations on the binding of biotin-labeled LPL to MPM. Binding of biotin-labeled LPL to
MPM was performed in the original RPMI-1640 medium containing 0.4 mM Ca
2 +
and 0.4 mM Mg
2 +
control, or in this medium with the addition of 1.34 mM EDTA, or with additional 1.4 mM Ca
2 +
or 2.6 mM Mg
2 +
by adding CaCl
2
· 2H
2
O and MgCl
2
· 6H
2
O. After washing the cells, Eu
3 +
-labeled streptavidin in RPMI-1640 medium was added to the cells and incubated for 30 min at 37°C. Fluores-
cence was then measured. Nonspecific binding with the omission of biotin-labeled LPL was subtracted from the total binding. Values
represent mean 9 SD of three experiments. P B 0.05 as compared with control.
Table 2 Deposition of cholesterol esters derived from native and differently oxidized LDL in MPM: effect of LPL and Ca
2+
concentrations
a
RPMI+LPL RPMI+1.4 mM Ca
2+
RPMI+LPL+1.4 mM Ca
2+
Synergistic effect RPMI control
2.33 9 0.11 356.9 2.24 9 0.09 339.2
LDL 4.62 9 0.25 805.9
0.51 9 0.0.02 0.56 109.8
oxLDL 1 h 0.72 9 0.03
1.85 9 0.12 156.9 5.93 9 0.53 723.6
9.82 9 0.52 597.9 2.76 383.3
0.96 9 0.05 54.8 5.95 9 0.62 859.7
oxLDL 3 h 8.98 9 0.93 1348.5
0.62 9 0.01 2.69 433.9
19.98 9 0.36 4.3 20.77 9 3.6 8.5
19.41 9 2.52 1.4 −
2.19 −11.4 19.15 9 0.52
oxLDL 24 h
a
Total and free cholesterol were measured after incubating MPM with native and differently oxidized LDL 100 mgml for 24 h in the original RPMI-1640 medium containing 0.4 mM Ca
2+
set as control, or in this medium with the addition of LPL 10 mgml, or with the addition of 1.4 mM Ca
2+
, or with the addition of both LPL 10 mgml and 1.4 mM Ca
2+
. Cholesterol ester was calculated by subtracting the value of free cholesterol from total cholesterol mg cholesterol estermg cell protein. Synergistic effects were calculated by subtracting the separately increased
cholesterol ester deposition in the presence of LPL and that in the presence of additional 1.4 mM Ca
2+
concentration from that in the presence of both LPL and additional 1.4 mM Ca
2+
. Values represent mean 9 SD of three experiments. Values in parentheses represent the percent increase compared with the controls, or the percent synergistic effect the last column of LPL and the increased Ca
2+
concentration. PB0.01;
PB0.05, as compared with the controls. Table 3
Effect of Ca
2+
and Mg
2+
concentrations on the binding of Eu
3+
-labeled native and differently oxidized LDL to LPL
a
RPMI control RPMI+1.4 mM Ca
2+
RPMI+EDTA RPMI+1.4 mM Mg
2+
0.28 9 0.01 2.16 9 0.15 671.4
0.11 9 0.01 −60.7 0.15 9 0.01 −46.4
LDL oxLDL 1 h
0.39 9 0.01 0.08 9 0.00 −79.5
3.52 9 0.21 890.2 0.17 9 0.00 −56.4
0.54 9 0.08 3.09 9 0.14 472.2
0.15 9 0.01 −72.2 0.26 9 0.01 −51.9
oxLDL 4 h 1.84 9 0.16
5.12 9 0.30 178.2 oxLDL 8 h
1.16 9 0.01 −36.9 1.06 9 0.02 −42.4
14.30 9 0.65 18.41 9 0.93 28.8
11.61 9 0.12 −18.8 12.04 9 0.08 −15.8
oxLDL 24 h
a
Microtitration plates were coated with LPL 1.5 mgwell at 4°C overnight. After blocking with BSA, Eu
3+
-labeled native and differently oxidized LDL in the original RPMI-1640 medium containing 0.4 mM Ca
2+
and 0.4 mM Mg
2+
set as control, or in this medium containing 1.34 mM EDTA, or with the addition of 1.4 mM Ca
2+
, or with the addition of 1.4 mM Mg
2+
, were incubated in the wells at 37°C for 1 h. After washing, the fluorescence was measured. Values are expressed as ng LDL proteinwell and represent mean 9 SD of three experiments. Values in
parentheses represent the percent increase or decrease − compared with the respective controls. PB
.05, PB0.01 as compared with the controls.
synergistically increased the accumulation in MPM of cholesterol esters derived from native, mildly or moder-
ately oxidized LDL Table 2. This synergistic effect was more evident for mildly and moderately oxidized
LDL, which is in agreement with the results from cell binding studies Table 1, while it was not observed
when extensively oxidized LDL 24 h oxidized LDL was used.
3
.
4
. Effect of Ca
2 +
on the binding of Eu
3 +
-labeled nati6e and oxidized LDL to LPL
Microtitration plates were coated with LPL, and the binding of Eu
3 +
-labeled native and oxidized LDL to LPL in the presence of different concentrations of
Ca
2 +
and Mg
2 +
was studied Table 3. The binding of Eu
3 +
-labeled native and oxidized LDL to LPL de- creased when EDTA was added to the medium to
chelate the divalent cations, and was enhanced with the increase of the Ca
2 +
concentration. The percent in- creased binding was more evident for native LDL and
mildly or moderately oxidized LDL than heavily oxi- dized LDL. In contrast, the addition of Mg
2 +
could not increase, but decreased the binding of native and
oxidized LDL to LPL. The results were similar when Tris – HCl buffer in stead of RPMI-1640 medium was
used not shown.
4. Discussion
