PLA2 stimulation of
Na+/H+
antiport and proliferation in rat aortic smooth muscle
cells
S.
Rufini1,
P.
De
Vito1,
N.
Balestro1,
M.
Pescatori1,
P.
Luly, and
S.
Incerpi2
1 Department of Biology,
University of Rome "Tor Vergata," 00133 Rome; and
2 Department of Biology,
University of Rome 3, 00146 Rome, Italy
 |
ABSTRACT |
The proliferative properties and the ability to stimulate the
Na+/H+
antiport activity of a secretory phospholipase
A2 were studied in rat aortic
smooth muscle cells in culture. The requirement of the enzymatic
activity of phospholipase A2 to
elicit mitogenesis was assessed by the use of ammodytin L, a
Ser49 phospholipase
A2 from the venom of
Vipera ammodytes, devoid of hydrolytic
activity. We propose that the proliferative effect is mediated by the
same transduction pathway for both proteins. In particular,
1) both secretory phospholipase
A2 and ammodytin L stimulated
thymidine incorporation in a dose-dependent manner; 2) both proteins affected the cell
cycle, as assessed by cell growth and fluorescence-activated cell
sorting experiments; 3) both
phospholipase A2 and ammodytin L
increased intracellular pH, a permissive factor for cell proliferation,
through activation of the
Na+/H+
antiport; 4) ammodytin L was able to
displace the 125I-labeled
phospholipase A2 from specific
binding sites in a concentration range consistent with that capable of
eliciting a cellular response; and
5) the inhibition by heparin was
similar for both proteins, taking into account the ratio of heparin to
protein. In conclusion, the enzymatic activity of phospholipase
A2 is not required for the
stimulation of mitogenesis. The inhibitory effect of heparin combined
with its therapeutic potential could help to clarify the role of
phospholipase A2 in the
pathogenesis of several preinflammatory situations.
phospholipases A2; snake
venom; ammodytin L; Na+/H+
antiport; growth factors; heparin
 |
INTRODUCTION |
SECRETORY (s) phospholipases
A2
(PLA2; EC 3.1.1.4) are
Ca2+-dependent enzymes that
specifically hydrolyze the two-ester bond of
1,2-diacyl-3-sn-phosphoglycerides to
give free fatty acids and lysophospholipids, precursors of potent
mediators of inflammation. Considered for a long time to exert a merely
digestive function, sPLA2 are
found in many mammalian tissues as well as in the venomous fluid of a
variety of animals (8). Two types of
sPLA2 have been identified so far
in mammalian circulating fluids [type I pancreatic
sPLA2
(psPLA2) and type II
sPLA2], but very little is
still known about their physiological role or mechanism of action (5,
21). In the past years, more and more pieces of evidence have been
produced describing biological effects of
sPLA2 challenge on different
cellular models in vitro (8, 11, 18). It has been proposed that many of
these effects can be explained by the generation of bioactive lipids
consequent to the enzymatic hydrolysis of membrane phospholipids on
target cells. Nevertheless, several pharmacological effects of
sPLA2 do not seem to involve their
hydrolytic activity (12, 13, 38). Binding and cross-linking experiments
with two phospholipases purified from the venom of taipan snake, OS1
and OS2, allowed Lambeau and colleagues (25, 26) to identify two
different sPLA2 membrane receptor
types. Neuronal-type receptor is the preferential target of neurotoxic sPLA2, including OS2 and bee venom
sPLA2 (25, 32). The second type
was originally isolated from rabbit skeletal muscle membranes and is
therefore referred to as muscular receptor (M type). M-type receptor
binds with high affinity various snake venom phospholipases as well as
pancreatic and inflammatory sPLA2,
suggesting that mammalian sPLA2
are probably natural ligands of this receptor (17, 23). The
identification of receptors for
sPLA2 opens up the possibility
that the biological properties of these proteins could be at least
partially ascribed to the activation of a receptor-mediated signal
transduction pathway (7, 14, 32). Many biological effects of type I
psPLA2 can be mimicked by several
snake venom type II sPLA2,
suggesting a common mechanism of action (13, 38).
sPLA2 receptor-mediated effects
described so far include the stimulation of cell proliferation in
several cell types (11), vascular smooth muscle contraction (41), and
chemokinesis (18). It is noteworthy that both
psPLA2 and a type II
sPLA2 from Naja naja venom were able to stimulate extracellular matrix
invasion by cancer cells, and this was demonstrated to require
detectable levels of expression of the high-affinity receptor (22). To further investigate the role of phospholipase activity in the pathophysiology of sPLA2 action on
target cells, we decided to compare the ability of ammodytin L (AMDL),
an enzymatically inactive snake venom type II
sPLA2, with type I
psPLA2 in inducing cellular response in rat aortic smooth muscle cells (RASMC), which have been
previously reported to be reactive to
psPLA2 stimulation (11). AMDL is a
myotoxic component of the venom of the snake Vipera
ammodytes. It belongs to the class of snake venom
PLA2-like proteins, which, on the
basis of their primary structure, strongly resemble type II
sPLA2 toxins but are devoid of
hydrolytic activity (20, 29). These proteins possess unique deviations
from the classical sequence of active enzymes, including the
replacement with Lys or Ser of
Asp49, a key residue that
coordinates a Ca2+ in the active
site (44). AMDL causes a selective and dramatic degeneration of the
skeletal muscle in vivo (3), whereas it displays complex
pharmacological behavior in vitro (15, 38). Concentrations of AMDL
capable of destroying differentiated myotubes in culture do not affect
other cell types like lymphocytes, erythrocytes, and platelets (15).
Interestingly, several cell lines in culture, such as fibroblasts and
human neuroblastoma, respond to AMDL challenge by increasing their
thymidine incorporation in analogy with the already mentioned
proliferative effect of psPLA2 in
vascular smooth muscle cells and synovial cells (1, 38). This body of
evidence together with the strong sequence homology of
psPLA2 and AMDL prompted us to
verify the possibility that psPLA2
and AMDL act through a common signal transduction pathway to assess the
role of hydrolytic activity on the proliferative response of
psPLA2. Our data show that
1)
psPLA2 stimulates cell
proliferation and the
Na+/H+
antiport activity, resulting in an increase of intracellular pH
(pHi), a permissive factor for cell proliferation and
2) the effect of
psPLA2 is fully mimicked by AMDL,
indicating that the hydrolytic activity is not a requirement for the
proliferative response of psPLA2.
The effect of psPLA2 and AMDL on
cell proliferation and pHi is
elicited through the binding to the same receptor and is inhibited by
heparin: the pathophysiological implications of this finding are also discussed.
 |
EXPERIMENTAL PROCEDURES |
Materials. AMDL was a gift of Dr. F. Gubensek (Jozef Stefan Institute, Ljubljana, Slovenia) and was purified
as described (24). Tissue culture medium, FCS, and sterile materials
were supplied by Flow Laboratories (Irvine, UK);
[3H]thymidine and
Na125I (carrier free) were
supplied by Amersham (Amersham, Bucks, UK); 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein
(BCECF)-AM was obtained from Molecular Probes (Eugene, OR). All other
chemicals were from Sigma (St. Louis, MO).
Cell culture. RASMC were prepared from
the explants of the thoracic aorta of 15-wk-old male Wistar rats by the
method of Ross (36). Cells were cultivated in 25-mm plastic tissue
culture flasks and were grown in DMEM supplemented with 10% FCS, 100 µg/ml streptomycin, and 100 U/ml penicillin in a humidified
atmosphere of 5% CO2 and 95% air
at 37°C. RASM cells, harvested one time per week by 0.25%
trypsin-0.02% EDTA and refed every other day, were used as confluent
monolayers after 6-8 days at passage level 4-9.
Analysis of DNA synthesis.
[3H]thymidine
incorporation was used to measure the mitogenic response. DNA synthesis
experiments were performed by incubating the cell monolayers in the
presence of AMDL. After incubation, the cells were pulsed with 1 µCi/ml of
[3H]thymidine and
incubated for an additional 3 h. After trypsin treatment for 3 min,
cells were harvested by centrifugation and treated with 5% TCA at
4°C for 30 min. The TCA-insoluble fraction was resuspended in 0.1%
SDS in 200 mM NaOH, and the samples were counted on a liquid
scintillation counter (LKB 1500). For cell proliferative assays, cells
were seeded and grown in 30-mm dishes in DMEM supplemented as reported
above and in the presence or absence of different effectors. For cell
counting, cells were harvested every 24 h by trypsinization and
centrifugation and then counted in a Thoma chamber. Each experiment was
carried out in triplicate.
Fluorescence-activated cell sorter experiments.
Cells washed in PBS were centrifuged at 200 g for 5 min. The pellet resuspended in
100 µl PBS was fixed in 70% (vol/vol) ethanol by adding on ice 900 µl of cold (
20°C) ethanol and was incubated on ice for at
least 30 min. After centrifugation, the pellet was washed one time in
1.5 ml PBS at room temperature and resuspended in 1 ml of DNA staining
solution (200 µg propidium iodide + 2 mg RNase A in 10 ml of PBS).
After 30 min at room temperature in the dark, DNA content of the cells
was assessed using a FACScan Flow Cytometer (Becton-Dickinson);
fluorescence was measured between 565 and 605 nm. The data were
acquired and analyzed by the Lysis II program (Becton-Dickinson).
pHi measurements.
For the experiments of fluorescence assays, cells were grown in chamber
slides (Lab-Tek; Nunc, Naperville, IL) and used at confluency no less
than 1 wk after plating. Before the experiment, cells were rendered
quiescent by serum deprivation for 24 h.
pHi was measured by the
fluorescent pHi indicator
BCECF-AM. To rule out the contribution of
HCO
3-dependent transport mechanisms
(16), all experiments were carried out in
HCO
3-free buffer with the following
composition (in mM): 135 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 10 glucose, and 20 HEPES,
pH 7.3. This buffer (designated as
Na+ buffer) was used for
incubation with the fluorescent probe and for the determination of
pHi, unless otherwise stated; the
cells incubated in this buffer were considered virtually depleted of HCO
3.
Solutions containing
NH3/NH+4
were prepared from the above buffer, replacing 20 mM NaCl with 20 mM
NH4Cl. During exposure to
NH4Cl, external
Na+ was routinely replaced by
equimolar choline chloride concentrations to keep the antiport
quiescent. Incubation with the fluorescent dye was carried out as
follows: cells were washed two times with Na+ buffer and were thus
considered HCO
3 free. Next, cells were
incubated in Na+ buffer with the
fluorescent dye (1 mg/ml in DMSO) at the final concentration of 1 µg
BCECF/ml for 30 min at 37°C in the dark. After the incubation, the
medium containing the dye was eliminated, and the cells were washed two
times with the same buffer. The calibration curve was carried out as
reported previously (42) using the nigericin method in a high-potassium
medium, with the same composition as the
Na+ buffer, but equimolar KCl
substituted NaCl. The calibration curve was linear in the range of pH
6.5-7.5 (not shown).
Fluorescence was also measured under continuous magnetic stirring at a
controlled temperature (37°C) in a Perkin-Elmer LS-5 luminescence
spectrometer equipped with a chart recorder model R 100A, with
excitation and emission wavelengths of 500 and 530 nm, using 5- and
10-nm slits, respectively, for the two light pathways.
Fluorescence was also routinely measured at 450 nm excitation (at this
wavelength, the fluorescence is proportional to intracellular dye
concentration but is relatively pH insensitive), and the value did not
change >10% during the experimental period.
Determination of intrinsic buffering
capacity. The total intracellular buffering capacity
(
t) is defined as follows
In
the nominally HCO
3-free solutions used
in this study, the buffering capacity of
CO2
(
CO2) was
assumed to be negligible, and
t
was therefore taken to be equal to the intrinsic buffering capacity
(
i). The
i was determined by using the
NH+4 pulse technique, as previously described (34, 35) according to the formula
where
[NH+4]i
represents the change in concentration of intracellular
NH+4 after exposure to or removal of
extracellular NH3, and
pHi represents the
corresponding change in pHi. The
intracellular concentration of NH+4 during
the NH4Cl pulse was calculated as
previously reported (35) from the following equation
taking
in account that NH3 equilibrates
across the cell membrane (i.e.,
[NH3]i = extracellular NH3 concentration)
and that the pKa of
NH+4 (8.92) is the same intra- and extracellularly.
[NH4Cl] in the absence
of NH4Cl was assumed to be zero.
Iodination of PLA2.
Iodination of psPLA2 was carried
out by using a chloramine T method, as described by Hanasaki and Arita
(11). Briefly, Na125I (10 MBq) was
added to 10 µg of psPLA2 in 100 µl of PBS, pH 7.4, and the reaction was initiated by adding 10 µl
of chloramine T (2 mg/ml). After 1 min of incubation at room
temperature, the reaction was terminated by adding 10 µl of 35 mM
-mercaptoethanol. After adding 20 µl of BSA (50 mg/ml) and 20 µl
of KI (100 mg/ml), the reaction mixture was loaded on a G-10 column,
and 125I-labeled
psPLA2 was eluted in PBS
containing 5 mg/ml BSA. The specific radioactivity obtained was 1,200 counts · min
1
(cpm) · pmol
1.
Binding experiments. Binding
experiments were carried out at 20°C in 300 µl of a buffer
consisting of 135 mM NaCl, 5 mM KCl, 0.1 mM
CaCl2, 10 mM glucose, 20 mM Tris,
pH 7.4, and 0.1% BSA. Confluent RASM cells were grown in 24-multiwell
(~5 × 105 cells/well)
culture plates (Corning), and, before the experiments, the cells were
washed two times with the above buffer and incubated with the
radiolabeled ligand in the presence and absence of unlabeled competitors. Nonspecific binding is defined as the difference between
the binding in the presence and absence of 1,500-fold molar excess of
unlabeled psPLA2. After 1 h, the
medium was removed, and cells were rapidly washed two times and then
lysed with 20 mM NaOH in 0.1% SDS. Radioactivity was assayed by a
gamma counter (5500 gamma; Beckman) with an efficiency of 80%.
 |
RESULTS |
RASM cells have been reported to respond to pancreatic
psPLA2 challenge with a
substantial increase on proliferation rate over untreated controls
(11). Our experiments show that AMDL, an
sPLA2-like snake myotoxin, is able
to induce an analogous response, even though it does not possess any
PLA2 activity. Pancreatic psPLA2 was 500 times more
effective than AMDL in stimulating cell proliferation, as assessed
either by cell count or
[3H]thymidine
incorporation. RASMC were incubated with increasing concentrations of
psPLA2 or AMDL for 48 h, pulsed
with [3H]thymidine,
and counted for radioactivity incorporation. Mean values from three
independent experiments (cpm × 10
3/well) are reported in
Fig. 1. Both proteins were able to
stimulate [3H]thymidine
incorporation over a two-log concentration range, although with
different EC50 values (AMDL
EC50 2.5 µg/ml,
psPLA2 EC50 5 ng/ml). Cell proliferation
rates, in treated RASMC and controls, were also measured by direct cell
count. As shown in Fig. 2, in experimental
samples, the cell number significantly increases over control values at
a concentration of 13 ng/ml for psPLA2 and 7 µg/ml for AMDL.
RASM cells (105/well) were grown
in the presence of either AMDL or
psPLA2, at the above
mentioned-concentration, and every 24 h cells were harvested and
counted. After a 48-h exposure to
sPLA2, we found a significant increase in cell number in treated samples. After 96 h, the treated cell number was increased by ~50% with respect to the control. In addition, sPLA2-treated RASM cells showed reduced
confluence-induced growth inhibition.

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 1.
Effect of ammodytin L (AMDL) and pancreatic (p) secretory (s)
phospholipase A2
(PLA2) on
[3H]thymidine
incorporation in rat aortic smooth muscle (RASM) cells. AMDL ( ) or
psPLA2 ( ) were added to
subconfluent RASM cells at indicated concentrations. After 48 h, cells
were pulsed with
[3H]thymidine and
processed as described in EXPERIMENTAL PROCEDURES. Results
are means ± SD of 3 independent experiments carried out in
triplicate. Brackets denote concentration; cpm, counts/min.
|
|

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 2.
Effect of AMDL and psPLA2 on RASM
cell growth rate. RASM cells were seeded in absence or presence of 13 ng/ml of psPLA2 or 7 µg/ml of
AMDL, and at indicated time cells were harvested and counted as
described in EXPERIMENTAL PROCEDURES. Results are means ± SD of 3 independent experiments carried out in triplicate.
|
|
In nonsyncronized cell cultures, the relative fraction of cells in
different stages of the cell cycle is a function of culture proliferation rate. FACScan DNA analysis (Fig.
3) shows that, in control cultures, ~63
and 29% of cells were in G1 and S
plus G2/M phase, respectively.
After 24 h of AMDL (7 µg/ml) treatment, the fraction of cells in S
plus G2/M phase increased to 41%
with compensatory reduction in the number of cells in
G1 phase (48%). Similar results were obtained with
psPLA2 (data not shown).

View larger version (9K):
[in this window]
[in a new window]
|
Fig. 3.
Typical DNA histograms of RASM cells subjected or not subjected to
long-term treatment with AMDL. Cells were incubated for 24 h with
vehicle (A) or 7 µg/ml AMDL
(B), stained with propidium iodide,
and analyzed by flow cytometry as described in EXPERIMENTAL
PROCEDURES. Axes represent 580 nm fluorescence emission by
propidium iodide (x) and number of
events (y).
G1, S, and
G2/M indicate the different phases
of the cycle.
|
|
The role of the growth factors in the mitogenic response of RASMC to
psPLA2 or AMDL stimulation was
also investigated. Serum or growth factors are essential for the
mitogenic action of sPLA2. As
indicated by
[3H]thymidine
incorporation experiments (Fig. 4), in
serum-free conditions, sPLA2
failed to show any proliferative effect on RASMC. The effect of FCS was
reproduced by epidermal growth factor (EGF) in a concentration between
10
9 and
10
7 M and to a lesser
extent by high insulin concentrations
(10
7 to
10
6 M; see Fig. 4).

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 4.
Action of serum and growth factors on AMDL and
psPLA2 effect on
[3H]thymidine
incorporation in RASM cells. Cells were grown in DMEM in presence of
serum (10%) or insulin (Ins;
10 7 M) or epidermal growth
factor (EGF; 10 8 M). AMDL
(7 µg/ml) or psPLA2 (13 ng/ml)
was added to the subconfluent RASM cells, and after 48 h cells were
pulsed with
[3H]thymidine and
processed as described in EXPERIMENTAL PROCEDURES. Results
are means ± SD of 3 independent experiments carried out in
triplicate.
|
|
The
Na+/H+
antiport is a plasma membrane protein that exchanges
Na+ and
H+ according to concentration
gradient. Besides the housekeeping function in the regulation of
pHi and cell volume, the
Na+/H+
antiport is considered to play a regulative role in cell
physiology (45). Many hormones and growth factors have been reported to be able to modulate pHi through activation of the
Na+/H+ antiport (4, 16, 45). We investigated
whether AMDL and psPLA2 could behave toward RASM cells as
growth factors also in stimulating Na+/H+
antiport activity.
Analysis of pHi at steady state
indicates that both proteins were able to induce a significant increase
in RASMC pHi in a concentration-dependent manner. Maximal activation of
Na+/H+
exchanger was reached by AMDL at 7-10 µg/ml and by
psPLA2 at 15 ng/ml, with an
increase of pHi over the basal
value after 15-min incubation time of 0.23 ± 0.03 (mean ± SD;
n = 4) for AMDL and 0.18 ± 0.04 (mean ± SD; n = 3) for
psPLA2 (Fig.
5).

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 5.
Effect of AMDL and psPLA2 on
steady-state intracellular pH
(pHi) in RASM cells. Results are
reported as change of pHi
( pHi)/15 min over basal value
after addition to confluent RASM cells of the indicated concentrations
of AMDL ( ) or psPLA2 ( ) and
are means ± SD of 3-6 separate experiments.
|
|
The time course of pHi recording
in the presence of AMDL and psPLA2
at different concentrations together with the effect of the derivative
of amiloride [ethylisopropyl amiloride (EIPA); 10 µM], a
specific inhibitor of the
Na+/H+
antiport, are reported in Fig. 6. To
analyze the effect of AMDL (7 µg/ml) and
psPLA2 (15 ng/ml) in conditions of
maximal
Na+/H+
antiport activation, we carried out experiments of acid load with
ammonium chloride (20 mM). Both AMDL and psPLA2 stimulated the Na+/H+ antiport, both as to the rate of
recovery (Fig.
7A) and
pH set point (Fig. 7B).
Regression lines computed from experimental values reported in Fig.
7B show that both rate of recovery and
pH set point in RASMC were increased by
sPLA2 stimulation. The slope of
the lines was not significantly different, whereas the intercept with
the x-axis was 7.03, 7.13, and 7.17 for the control, AMDL-, and
psPLA2-treated cells, respectively
(Fig. 7B).

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 6.
Time course of AMDL and psPLA2
effect on pHi at steady state in
RASM cells. Traces are representative of at least 3 similar
experiments. Inhibitory effect of ethylisopropyl amiloride (EIPA; 10 µM) is also shown for both AMDL and
psPLA2 at the highest
concentrations. Arrows indicate the addition of AMDL or
psPLA2.
|
|


View larger version (30K):
[in this window]
[in a new window]
|
Fig. 7.
Recorder tracing of acid load, obtained with
NH3/NH+4
pulse and pHi recovery in RASM
cells: effect of AMDL and psPLA2.
A: traces are representative
experiments whose means are reported as regression lines in
B. Protein (7 µg/ml AMDL and 13 ng/ml psPLA2) addition is
indicated by the arrow. B:
computer-generated regression lines represent means from 4-6
different experiments. , Control; , AMDL; ,
psPLA2.
|
|
From data reported in Table 1, the net
efflux of acid (i.e., acid extrusion rate, J) can be
calculated. J is the product of
and the rate of
pHi recovery (36). In the nominal
absence of HCO
3, the
value is the
intrinsic buffer power that at pHi
6.8 is 38 ± 9 mM/pH unit (mean ± SD;
n = 3), well in agreement with
previously published data (16). J was evaluated as 1,140 × 10
4, 2,145 × 10
4, and 3,300 × 10
4 mM/s at
pHi 6.8 for control, AMDL-, and
psPLA2-treated cells, respectively.
View this table:
[in this window]
[in a new window]
|
Table 1.
Effect of AMDL and psPLA2 on
pHi, buffering power, and rate of
pHi recovery from an acid load in RASM cells
|
|
Heparin has been shown to exert protective effects toward several
PLA2 and
PLA2-like myotoxins, and it is
also known to interfere with a wide variety of biological proteins (28,
30). Heparin was able to inhibit the
sPLA2-mediated increase in
thymidine incorporation (Fig. 8). The data
show that the heparin concentration capable of preventing the
sPLA2 activity was three orders of
magnitude lower than the concentration required to block AMDL. These
data are consistent with the hypothesis that the target of the heparin is the added protein (i.e., psPLA2
and AMDL) rather than a molecule involved in the binding and/or in the
transduction pathway.

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 8.
Heparin effect on AMDL and psPLA2
action on RASM cells. RASM cells were briefly incubated with heparin at
the indicated concentrations and then stimulated with AMDL (7 µg/ml)
or psPLA2 (13 ng/ml). Cells were
pulsed with
[3H]thymidine and
processed as described in EXPERIMENTAL PROCEDURES. Results
are means ± SD of 3 independent experiments carried out in
triplicate.
|
|
The strong similarity between AMDL and
psPLA2 effects on cell growth and
pHi appeared to be very puzzling,
and we hypothesized that the two proteins may bind to the same
receptor. Therefore, binding and competition experiments were carried
out by using 125I-psPLA2
and increasing AMDL concentrations. Preliminary experiments indicated
that maximum binding for the
125I-psPLA2
was achieved at 20°C in 120 min and was stable for at least 2 h
(not shown).
125I-psPLA2
binding at equilibrium to RASM cells gave a straight line, indicating
the presence of a single class of binding sites and, from Scatchard
analysis, an equilibrium binding constant (Kd) value of 4 nM, well in agreement with data reported by Hanasaki and Arita (11) for
the same cells. To investigate whether
psPLA2 and AMDL bind to the same
receptor in the RASM cells, our first attempt was to study the
Kd of AMDL to
compare its kinetic values with the those observed for
psPLA2. We failed to obtain
reliable values, due to the high level of nonspecific binding showed by this class of proteins. Basic myotoxins seem to possess a very high
affinity for negatively charged surfaces, such as plastic for cell
culture and glass. For this reason, as a strategy to approach the
problem, we decided to perform competition experiments using labeled
psPLA2 and increasing
concentrations of AMDL. The relative inhibitory effects of various
concentrations of unlabeled psPLA2
and AMDL on
125I-psPLA2
equilibrium binding to RASM cells are shown in Fig.
9A. The
concentration of psPLA2 that
inhibits one-half of the
125I-psPLA2
specific binding
(K0.5) was
10
9 M. AMDL also inhibited
125I-psPLA2
specific binding to RASM cells but with a
K0.5 more than two orders of magnitude (2 × 10
7 M), indicating a
difference in affinity between
psPLA2 and AMDL. The same strategy
was used to investigate the effect of heparin on the
125I-psPLA2
binding to the RASM cells. Figure 9B
indicates that heparin prevents
125I-psPLA2
binding to the cells in a dose-dependent manner. An apparent K0.5 was raised
at a heparin concentration two to three orders of magnitude higher
(molar ratio) than the phospholipase concentration. In these
experiments, it is important to take into account that the
125I-psPLA2
binding displacement by heparin did not possess the same significance
of the data obtained with phospholipases. In fact, heparin likely binds
to several "high-affinity" binding sites on the surface of the
cells, and an exact calculation of its
K0.5 on
125I-psPLA2
binding could be artifactual. Our results purely indicate that the
heparin able to inhibit the
125I-psPLA2
binding to RASM cells is, as expected, close to the concentration that
inhibits the physiological effect of phospholipases.


View larger version (30K):
[in this window]
[in a new window]
|
Fig. 9.
Competition of
125I-psPLA2
specific binding and unlabeled
psPLA2 or AMDL and heparin.
125I-psPLA2
(4 nM) was incubated with RASM cells in the presence of increasing
concentration of proteins (A) or
heparin (B). Results are expressed
as percentage of the maximal specific binding measured in the absence
of competitor. One-hundred percent corresponds to a specifically bound
concentration of 4 nM of
125I-psPLA2;
0% is the
125I-psPLA2
binding measured in the presence of 1,500-fold excess unlabeled
psPLA2. Values shown are
representative of at least 3 experiments.
|
|
 |
DISCUSSION |
This study shows that both psPLA2
and AMDL can stimulate RASMC proliferation in culture and that this
effect is preceded by an increase of
pHi, dependent on the activation
of the
Na+/H+
antiport. We propose that the proliferative effect is mediated by the
same transduction pathway for both proteins.
To support this statement, we demonstrate that
1) both
psPLA2 and AMDL stimulate
thymidine incorporation by RASMC in a dose-dependent manner, although a
500-fold higher AMDL concentration is required to elicit an identical
response; 2) AMDL was able to
displace the
125I-PLA2
from specific binding sites in a concentration range consistent with
that capable of eliciting a cellular response;
3) cell growth and FACScan
experiments indicate that psPLA2
and AMDL affect cell cycle control;
4) the presence of FCS and/or growth
factors is required by both proteins to exert their stimulatory effect
on DNA synthesis; 5) the relative
concentrations of psPLA2 or AMDL necessary for the activation of
Na+/H+
exchange activity are similar to the concentrations required to
stimulate RASM cell proliferation;
6) the time course of the effect of
psPLA2 and AMDL on RASMC
pHi is superimposable; and 7) the inhibition by heparin is
similar for both proteins, taking into account the ratio of heparin to protein.
Although there is compelling evidence for a central role of
sPLA2 in the pathogenesis of
several preinflammatory situations, very little is still known about
the molecular details of the cascade of events that follow the
interaction of psPLA2 with target cells (43). Different cellular models have been reported to react to
psPLA2 stimulation by
proliferating, likely via the activation of the mitogen-activated
protein kinase cascade (13). A direct involvement of
sPLA2 in proliferation and
migration of RASMC, after arterial injury, appears to be a pivotal
clinical problem in the management of atherosclerosis and angiogenesis
occurring in response to a variety of physiological or
pathophysiological stimuli (46). Autocrine mechanisms of production of
growth factors by RASMC, such as platelet-derived growth factor (PDGF),
EGF, and insulin-like growth factor I (IGF-I) acting synergically, are
responsible for RASMC proliferation after injury (37). When RASMC are
incubated with proinflammatory cytokines, such as tumor necrosis
factor, interleukin-1, or cAMP-elevating agents, gene expression of
sPLA2 is induced, and the newly
generated sPLA2 are found in the
extracellular medium (31).
Because many hydrolysis products of phospholipids by
sPLA2 (i.e., arachidonic acid,
lysophosphatidylcholine, and lysophosphatidic acid) are mediators of
the inflammatory response, the most obvious model for
sPLA2 action hypothesizes the
hydrolysis of plasma membrane phospholipids of target cells and the
consequent release of active lipids. Nevertheless, in living cells,
plasma membrane phospholipids are a very poor substrate for
sPLA2, and extensive phospholipid hydrolysis by exogenous sPLA2 has
been described only in microvesicles shed from activated cells and
membranes derived from cells undergoing apoptosis (2, 10).
An intriguing question arises about the role of
sPLA2 enzymatic properties in the
activation of this signal transduction pathway. Do
sPLA2, in the induction of their
proliferative response, behave just like receptor ligands, or, upon
binding on the receptor, do they hydrolyze cell membrane phospholipids
to produce bioactive lipid species? This point is still a matter of
debate. Arachidonic acid production has been demonstrated after
incubation of cultured cells with
sPLA2 (13), but the total absence
of oleate or unsaturated fatty acid from culture supernatant induced
Hernandez et al. (13) to imply the activation of a cytoplasmic
phospholipase that, unlike sPLA2, is known to be highly
selective for arachidonic acid-containing membrane phospholipids.
In this work, we provide evidence that AMDL, a
sPLA2-like snake toxin that does
not possess any hydrolytic activity, is able to trigger a mitogenic
response in RASM cells. The fact that phospholipase activity does not
seem to be required for sPLA2
stimulation of mitogenesis strongly supports the hypothesis that
sPLA2 acts as pure ligand by
activating a cell membrane receptor.
sPLA2-like toxins, in this
context, are natural sPLA2 mutants
and can represent a valuable tool for unraveling the pharmacological
properties of this class of molecules.
Binding experiments on RASM cells have shown that these cells do
express an sPLA2 receptor whose
relative molecular mass and sPLA2
binding affinity indicate that it belongs to the M-type receptor class
(11). A good correlation exists between cell responsiveness and the
expression of the M-type receptor for
sPLA2, a 180-kDa glycoprotein that
shows strong homology with the macrophage mannose-binding receptor (5).
Kd values of
sPLA2 binding to the M-type
receptor are generally in agreement with
EC50 values established for
sPLA2 biological effects. Binding
to the M-type receptor has been reported to be a first step in the
sPLA2-induced mitogenic response,
but further investigations are needed to clarify how the signal is
transduced across the cell membrane and how it modulates cell
cycle. Our data confirm the existence of a high-affinity binding site for psPLA2 with a
Kd close to
2-4 nM. Inhibition experiments demonstrate that AMDL totally
inhibits the
125I-psPLA2
specific binding to RASM cells, suggesting a competition for the same
binding site. The
K0.5 values of
AMDL indicate that this protein possesses an affinity for the
psPLA2 receptor of two orders of
magnitude lower with respect to
psPLA2. As specified in
RESULTS, we did not obtain reliable values of AMDL binding due to the presence of high nonspecific binding. The problem is only
partially resolved by experiments of
125I-psPLA2
displacement, probably because only a fraction, likely dependent on the
AMDL concentration, was available to compete with the
psPLA2. In other words, our
experiments only indicate a competition of the two proteins for the
same receptor, but the K0.5 values
cannot be taken as absolute values. Despite all these problems, these
data are in good agreement with the different effectiveness of AMDL
with respect to psPLA2 in
triggering a cellular response (i.e., pH increase and a comitogen
effect). Recently, Cupillard et al. (6) published a paper in which it
was proven that both phospholipase groups, i.e., group I
(psPLA2) and group II (snake
venom PLA2), can bind, even with
different affinity, to the 180-kDa M-type receptor. Interestingly,
Cupillard et al. (6) first reported that the affinity for the M-type
receptors shown by different classes of phospholipases varies depending on the animal source of the receptor. This finding can
help to clarify a confusing situation existing in the literature (6).
Dose-response analysis of
[3H]thymidine
incorporation by AMDL- or
psPLA2-treated RASM cells shows
how AMDL stimulates DNA synthesis to the same extent as
psPLA2, but 500-fold higher
concentrations are required. Our results are in agreement with the data
reported by Lambeau and co-workers (24) that different mutants of
recombinant psPLA2 show different
affinity for the receptor. psPLA2
mutants with Asp49-Lys amino acid
substitution display a
Kd for M-type
receptor two orders of magnitude lower than the wild-type
sPLA2. Because AMDL carry an
Asp49-Ser amino acid substitution
with respect to the sequence of active sPLA2, we suggest that the
500-fold difference between AMDL and sPLA2 in the relative potency in
stimulating RASMC proliferation could be explained as the effect of a
500-fold difference in the binding affinity.
On the basis of our results, the transduction pathway involves an
increase in pHi, due to activation
of the
Na+/H+
antiport, a permissive factor for cell proliferation (35). In fact,
both psPLA2 and AMDL were able to
induce an increase in pHi at
equilibrium and under conditions of best activation for the antiport,
after an acid load with ammonium chloride. The specificity of the
effect is assessed by the inhibition of the antiport by the derivative
of amiloride, EIPA. The effect on
pHi is dose dependent, and
pHi is in the same range as the one reported for
hormones and growth factors (35).
The buffering capacity (
) and the rate of recovery
(dpHi/dt)
show only a trend to an increase in
psPLA2- and AMDL-treated cells,
but J, the net efflux acid, increases in treated cells. The
increased J, in turn, could activate the
Na+/Ca2+
exchanger, due to the increased intracellular
Na+, allowing a massive influx of
Ca2+ from extracellular medium. In
the pathological condition in which sPLA2 is greatly increased, as in
the ischemic condition, this Ca2+
overload could give rise to cell death (9, 27).
Furthermore, our data show that
psPLA2 are weak mitogens for RASMC
but greatly increase the proliferative activity of serum. Both EGF and
insulin (at concentration
>10
7 M) can substitute
for serum as a comitogen factor. RASMC proliferation can be stimulated
by a variety of growth factors, and relevant information is available
on the synergy between growth factors (37, 46). For example, weak
mitogens such as IGF-I increase the ability of other mitogens such as
PDGF, fibroblast growth factor, and EGF (33), and insulin is an
important comitogen for ANG II and EGF (19). However, the basis of this
synergy between growth factors is not well understood. A hypothesis
could be that sPLA2 stimulates the
expression of the comitogen receptors or that the increase of
intracellular Ca2+ and/or pH
amplifies the mitogen effect.
Heparin has been reported to inhibit cell growth and
Na+/H+
exchange in vascular smooth muscle cells (7). The fact that heparin inhibits activity of both psPLA2-
and AMDL-induced cell proliferation to the same extent provides further
support for the hypothesis of a common mechanism of action of these two
proteins. Our competition experiments on
125I-psPLA2
binding clearly show that heparin inhibits the binding of the
phospholipase to the RASM cells. It is likely that the inhibitory
effect of heparin on
psPLA2-induced cell growth is due
to a direct effect of the glycan on the phospholipase binding and not
to the inhibition of its transduction process. This statement is in
good agreement with the recent observation that
PLA2 binds, as several growth
factors, the cellular matrix of vascular cells and that heparin can
prevent this binding (40). On the basis of our data, we are not able to
demonstrate if heparin inhibits the
psPLA2 binding to its receptor by
a direct interaction with the phospholipase or with the receptor.
In any case, the relationship between heparin and different classes of
PLA2 is very puzzling. Lomonte et
al. (28) have shown that heparin blocks the cytolytic effect of the
snake venom myotoxin II, probably through the binding with the fragment
119-125 COOH-terminal. Analogously, recombinant
sPLA2, lacking a Lys residue in
the COOH-terminal, is unable to bind both heparin and cells in culture
but retains its hydrolytic activity on artificial phospholipid
membranes (30). The same terminal Lys-mutated
sPLA2 bind preapoptotic cells,
which are known to expose acidic phospholipids on the external surface of the plasma membrane. Furthermore, a previous paper from our laboratory has shown that the
PLA2-like myotoxins can penetrate in the hydrophobic core of artificial bilayers only if acidic lipids
are present (39). Finally, a common pathway between toxic, pancreatic,
and secreted PLA2 seems to be the
binding to a "negatively charged compartment" on the surface of
the cell membrane. Heparin could inhibit the different physiological
and pharmacological sPLA2 effects
through the same mechanism. More focused experiments, using purified
heparin molecular species, must be carried out to identify the
polysaccharide sequence that binds with high affinity to the
sPLA2. These data could be of
enormous interest considering the therapeutic potential of heparin, a
drug whose anti-inflammatory and antiatherosclerotic role is so far
stated but poorly understood.
 |
ACKNOWLEDGEMENTS |
We thank Dr. R. Musanti from the Laboratory of Cellular
Pharmacology of Atherosclerosis, Pharmacia, Nerviano, Milan, Italy, for
providing the RASM cells and Dr. Alessandra Amendola for the cytometric
analysis. We also thank Dr. Alfonso Grasso for fruitful discussion and
helpful suggestions.
 |
FOOTNOTES |
This study was supported by grants from the Italian Ministry of
University and Scientific-Technological Research.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: S. Rufini,
Dipartimento di Biologia, Universita di Roma "Tor Vergata," Via
della Ricerca Scientifica, 00133 Rome, Italy (E-mail:
rufini{at}uniroma2.it).
Received 10 September 1998; accepted in final form 3 June 1999.
 |
REFERENCES |
1.
Arita, H.,
K. Hanasaki,
T. Nakano,
S. Oka,
H. Teraoka,
and
K. Matsumoto.
Novel proliferative effect of phospholipase A2 in Swiss 3T3 cells via specific binding site.
J. Biol. Chem.
266:
19139-19141,
1991[Abstract/Free Full Text].
2.
Atsumi, G.-I.,
M. Murakami,
M. Tajima,
S. Shimbara,
N. Hara,
and
I. Kudo.
The perturbated membrane of cells undergoing apoptosis is susceptible to type II secretory phospholipase A2 to liberate arachidonic acid.
Biochim. Biophys. Acta
1349:
43-54,
1997[Medline].
3.
Bernardini, S.,
S. M. Cannata,
S. Filoni,
P. Luly,
and
S. Rufini.
Effect of ammodytin L from the venom of Vipera ammodytes on Xenopus laevis differentiated muscle fibers and regenerating limbs.
Toxicon
34:
81-90,
1996[Medline].
4.
Ciriolo, M. R.,
A. T. Palamara,
S. Incerpi,
E. Lafavia,
M. C. Bue',
P. De Vito,
E. Garaci,
and
G. Rotilio.
Loss of GSH, oxidative stress, and decrease of intracellular pH as sequential steps in viral infection.
J. Biol. Chem.
272:
2700-2708,
1997[Abstract/Free Full Text].
5.
Cupillard, L.,
K. Koumanov,
M.-G. Mattei,
M. Lazdunski,
and
G. Lambeau.
Cloning, chromosomal mapping, and expression of a novel human secretory phospholipase A2.
J. Biol. Chem.
272:
15745-15752,
1997[Abstract/Free Full Text].
6.
Cupillard, L.,
R. Mulherkar,
N. Gomez,
S. Kadam,
E. Valentin,
M. Lazdunski,
and
G. Lambeau.
Both group IB and group IIA secreted phospholipases A2 are natural ligands of the mouse 180kDa M-type receptor.
J. Biol. Chem.
274:
7043-7051,
1999[Abstract/Free Full Text].
7.
Dahlberg, C. G. W.,
B. T. Thompson,
P. M. Joseph,
H. G. Garg,
C. R. Spence,
D. A. Quinn,
J. V. Bonventre,
and
C. A. Hales.
Differential effect of three commercial heparins on Na/H exchange and growth of RASMC.
Am. J. Physiol.
270 (Lung Cell. Mol. Physiol. 14):
L260-L265,
1996[Abstract/Free Full Text].
8.
Dennis, E. A.
Diversity of group types, regulation and function of phospholipases A2.
J. Biol. Chem.
269:
13057-13060,
1994[Free Full Text].
9.
Fliegel, L.,
and
O. Frohlich.
The Na/H exchanger an update on structure, regulation and cardiac physiology.
Biochem. J.
296:
273-285,
1993[Medline].
10.
Fourcade, O.,
M.-F. Simons,
C. Viadé,
N. Rugani,
F. Leballe,
A. Ragab,
B. Fourni,
L. Sarda,
and
H. Chap.
Secretory phospholipase A2 generates the novel lipid mediator lysophosphatidic acid in membrane microvesicles shed from activated cells.
Cell
80:
919-927,
1995[Medline].
11.
Hanasaki, K.,
and
H. Arita.
Characterization of an high affinity binding site for pancreatic-type phospholipase A2 in the rat. Its cellular and tissue distribution.
J. Biol. Chem.
267:
6414-6420,
1992[Abstract/Free Full Text].
12.
Hanasaki, K.,
Y. Yokota,
J. Ishizaki,
T. Itoh,
and
H. Arita.
Resistance to endotoxic shock in phospholipase A2 receptor-deficient mice.
J. Biol. Chem.
272:
32792-32797,
1997[Abstract/Free Full Text].
13.
Hernandez, M.,
S. L. Burillo,
M. S. Crespo,
and
M. L. Nieto.
Secretory phospholipase A2 activates the cascade of mitogen-activated protein kinases and cytosolic phospholipase A2 in the human astrocytoma cell line 1321N1.
J. Biol. Chem.
273:
606-612,
1998[Abstract/Free Full Text].
14.
Higashino, K.-I.,
J. Ishizaki,
J. Kishino,
O. Ohara,
and
H. Arita.
Structural comparison of phospholipase-A2-binding regions in phospholipase-A2 receptors fom various mammals.
Eur. J. Biochem.
225:
375-382,
1994[Abstract].
15.
Incerpi, S.,
P. De Vito,
P. Luly,
and
S. Rufini.
Effect of ammodytin L from Vipera ammodytes on L6 cells from rat skeletal muscle.
Biochim. Biophys. Acta
1268:
137-142,
1995[Medline].
16.
Incerpi, S.,
S. Spagnuolo,
F. Terenzi,
and
S. Leoni.
EGF modulation of Na+/H+ antiport in rat hepatocytes: different sensitivity in adult and fetal cells.
Am. J. Physiol.
270 (Cell Physiol. 39):
C841-C847,
1996[Abstract/Free Full Text].
17.
Ishizaki, J.,
K. Hanasaki,
K.-I. Higashino,
J. Kishino,
N. Kikuchi,
O. Ohara,
and
H. Arita.
Molecular cloning of pancreatic group I phospholipase A2 receptor.
J. Biol. Chem.
269:
5897-5904,
1994[Abstract/Free Full Text].
18.
Kanemasa, T.,
K. Hanasaki,
and
H. Arita.
Migration of vascular smooth muscle cells by phospholipase A2 via specific binding sites.
Biochim. Biophys. Acta
1125:
210-214,
1992[Medline].
19.
Ko, Y.,
A. Sahinidis,
A. J. Wieczorek,
M. Appenheimer,
R. Dusing,
and
H. Vetter.
Insulin enhances angiotensin II induced DNA synthesis in vascular smooth muscle cells of the rat.
Clin. Invest. Med.
71:
379-382,
1993.
20.
Krizaj, I.,
A. L. Bieber,
A. Ritonja,
and
F. Gubensek.
The primary structure of ammodytin L, a myotoxic phospholipase A2 homologue from Vipera ammodytes venom.
Eur. J. Biochem.
202:
1165-1168,
1991[Abstract].
21.
Kudo, I.,
M. Murakami,
S. Hara,
and
K. Inoue.
Mammalian non-pancreatic phospholipases A2.
Biochim. Biophys. Acta
1170:
217-231,
1993[Medline].
22.
Kundu, G. C.,
and
A. B. Mukherjee.
Evidence that porcine pancreatic phospholipase A2 via its high affinity receptor stimulates extracellular matrix invasion by normal and cancer cells.
J. Biol. Chem.
272:
2346-2353,
1997[Abstract/Free Full Text].
23.
Lambeau, G.,
F. Ancian,
B. Barahanin,
and
M. Lazdunski.
Cloning and expression of a membrane receptor for secretory phospholipases A2.
J. Biol. Chem.
269:
1575-1578,
1994[Abstract/Free Full Text].
24.
Lambeau, G.,
P. Ancian,
J.-P. Nicolas,
H. W. Beiboer,
D. Moinier,
H. Verheij,
and
M. Lazdunski.
Structural elements of secretory phospholipases A2 in the binding of M-type receptor.
J. Biol. Chem.
270:
5534-5540,
1995[Abstract/Free Full Text].
25.
Lambeau, G.,
B. Barahanin,
H. Schweitz,
J. Qar,
and
M. Lazdunski.
Identification and properties of very high affinity brain membrane-binding sites for a neurotoxic phospholipase from the taipan venom.
J. Biol. Chem.
264:
11503-11510,
1989[Abstract/Free Full Text].
26.
Lambeau, G.,
A. Schmid-Alliana,
M. Lazdunski,
and
B. Barahanin.
Identification and purification of a very high affinity binding protein for toxic phospholipase A2 in skeletal muscle.
J. Biol. Chem.
265:
9526-9528,
1990[Abstract/Free Full Text].
27.
Lauritzen, I.,
C. Heurteaux,
and
M. Lazdunski.
Expression of group II phospholipase A2 in rat brain after severe forebrain ischemia and in endotoxic shock.
Brain Res.
651:
353-356,
1994[Medline].
28.
Lomonte, B.,
E. Moreno,
A. Tarkowski,
L. A. Hanson,
and
M. Maccarana.
Neutralizing interaction between heparins and myotoxin II, a lysine-49 phospholipase A2 from Bothrops asper snake venom. Identification of a heparin-binding and cytolytic toxin region by the use of synthetic peptides of molecular modelling.
J. Biol. Chem.
269:
29867-29873,
1994[Abstract/Free Full Text].
29.
Maraganore, J. M.,
G. Merutka,
W. Cho,
W. Welches,
F. J. Kzdy,
and
R. L. Heinrikson.
A new class of phospholipases A2 with lysine in place of aspartate 49. Functional consequences for calcium and substrate binding.
J. Biol. Chem.
259:
13839-13843,
1984[Abstract/Free Full Text].
30.
Murakami, M.,
Y. Nakatani,
and
I. Kudo.
Type II secretory phospholipase A2 associated with cell surfaces via C-terminal heparin-binding lysine residues augments stimulus-initiated delayed prostaglandin generation.
J. Biol. Chem.
271:
30041-30051,
1996[Abstract/Free Full Text].
31.
Nakano, T.,
O. Ohara,
H. Teraoka,
and
H. Arita.
Glucocorticoids suppress group II phospholipase A2 production by blocking mRNA synthesis and post-transcriptional expression.
J. Biol. Chem.
265:
12745-12748,
1990[Abstract/Free Full Text].
32.
Nicolas, J. P.,
Y. Lin,
G. Lambeau,
F. Ghomashchi,
M. Lazdunski,
and
M. H. Gelb.
Localization of structural elements of bee venom phospholipase A2 involved in N-type receptor binding and neurotoxicity.
J. Biol. Chem.
272:
7173-7181,
1997[Abstract/Free Full Text].
33.
Reape, T. J.,
J. M. Kanczler,
J. P. T. Ward,
and
C. R. Thomas.
IGF-I increases bFGF-induced mitogenesis and upregulates FGFR-1 in rabbit vascular smooth muscle cells of the rat.
Am. J. Physiol.
270 (Heart Circ. Physiol. 39):
H1141-H1148,
1996[Abstract/Free Full Text].
34.
Ricci, R.,
P. Baldini,
L. Bogetto,
P. De Vito,
A. Zannetti,
and
S. Incerpi.
Dual modulation of Na/H antiport by atrial natriuretic factor in rat aortic smooth muscle cells.
Am. J. Physiol.
273 (Cell Physiol. 42):
C643-C652,
1997[Abstract/Free Full Text].
35.
Roos, A.,
and
W. F. Boron.
Intracellular pH.
Physiol. Rev.
61:
296-434,
1981[Free Full Text].
36.
Ross, R.
The smooth muscle cells. In vivo synthesis of connective tissue proteins.
J. Cell Biol.
50:
172-186,
1971[Abstract/Free Full Text].
37.
Ross, R.
The pathogenesis of atherosclerosis: a perspective for the 1990s.
Nature
362:
801-809,
1993[Medline].
38.
Rufini, S.,
M. P. Cesaroni,
N. Balestro,
and
P. Luly.
Proliferative effect of ammodytin L from the venom of Vipera ammodytes on 208F rat fibroblasts in culture.
Biochem. J.
320:
467-472,
1996[Medline].
39.
Rufini, S.,
M. P. Cesaroni,
A. Desideri,
R. N. Farias,
F. Gubensek,
J. Gutierrez,
P. Luly,
R. Massoud,
R. Morero,
and
J. Z. Pedersen.
Ca2+-independent membrane leakage induced by phospholipase like myotoxins.
Biochemistry
31:
12424-12430,
1992[Medline].
40.
Sartipy, P.,
G. Bondjers,
and
E. Hurt-Camejo.
Phospholipase A2 type II binds to extracellular matrix biglycan.
Arterioscler. Thromb. Vasc. Biol.
18:
1934-1941,
1998[Abstract/Free Full Text].
41.
Sommers, C. D.,
J. L. Bobbitt,
K. G. Bemis,
and
D. W. Snyder.
Porcine pancreatic phospholipase A2-induced contraction of guinea pig lung pleural strips.
Eur. J. Pharmacol.
216:
87-96,
1992[Medline].
42.
Thomas, J. A.,
R. N. Buchsbaum,
A. Zimniak,
and
E. Racker.
Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ.
Biochemistry
18:
2210-2218,
1979[Medline].
43.
Vadas, P.,
J. Browning,
J. Edelson,
and
W. Pruzanski.
Extracellular phospholipase A2 expression and inflammation: the relationship with associated disease states.
J. Lipid Mediators
8:
1-30,
1993[Medline].
44.
Van den Bergh, C. J.,
A. J. Slotboom,
H. M. Verheij,
and
G. H. De Haas.
The role of Asp-49 and other conserved aminoacids in phospholipase A2 and their importance for enzymatic activity.
J. Cell. Biochem.
39:
379-390,
1989[Medline].
45.
Wakabayashi, S.,
M. Shigekawa,
and
J. Pouyssegur.
Molecular physiology of vertebrate Na/H exchangers.
Physiol. Rev.
77:
51-74,
1997[Abstract/Free Full Text].
46.
Wilcox, J. N.
Molecular biology insight into the causes and prevention of restenosis after arterial intervention.
Am. J. Cardiol.
72:
88E-95E,
1993[Medline].
Am J Physiol Cell Physiol 277(4):C814-C822
0002-9513/99 $5.00
Copyright © 1999 the American Physiological Society