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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (beta t) is defined as follows
&bgr;<SUB>t</SUB> = &bgr;<SUB>CO<SUB>2</SUB></SUB> + &bgr;<SUB>i</SUB>
In the nominally HCO-3-free solutions used in this study, the buffering capacity of CO2 (beta CO2) was assumed to be negligible, and beta t was therefore taken to be equal to the intrinsic buffering capacity (beta i). The beta i was determined by using the NH+4 pulse technique, as previously described (34, 35) according to the formula
&bgr;<SUB>i</SUB> = &Dgr;[NH<SUP>+</SUP><SUB>4</SUB>]<SUB>i</SUB>/&Dgr;pH<SUB>i</SUB>
where Delta [NH+4]i represents the change in concentration of intracellular NH+4 after exposure to or removal of extracellular NH3, and Delta 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
[NH<SUP>+</SUP><SUB>4</SUB>]<SUB>i</SUB>= [NH<SUB>3</SUB>]<SUB>i</SUB> × 10<SUP>8.92 − pH<SUB>i</SUB></SUP>
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 beta -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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


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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 (open circle ) 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.



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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).


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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).


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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).


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Fig. 5.   Effect of AMDL and psPLA2 on steady-state intracellular pH (pHi) in RASM cells. Results are reported as change of pHi (Delta pHi)/15 min over basal value after addition to confluent RASM cells of the indicated concentrations of AMDL (open circle ) 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).


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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.




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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. star , Control; open circle , 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 beta  and the rate of pHi recovery (36). In the nominal absence of HCO-3, the beta  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.

                              
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Table 1.   Effect of AMDL and psPLA2 on Delta 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.


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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.



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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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 Delta pHi is in the same range as the one reported for hormones and growth factors (35).

The buffering capacity (beta ) 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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Cell Physiol 277(4):C814-C822
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