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J Biol Chem, Vol. 274, Issue 40, 28134-28141, October 1, 1999

Inositol 1,4,5-Trisphosphate-independent Ca²⁺ Mobilization Triggered by a Lipid Factor Isolated from Vitreous Body^*

Jesus P. Camiña, Xesus Casabiell^§, and Felipe F. Casanueva^¶

From the Departments of  Medicine and ^§ Physiology, Cellular Endocrinology Laboratory, Compostela University School of Medicine and Complejo Hospitalario Universitario de Santiago, E-15780 Santiago de Compostela, Spain

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A complex phospholipid from bovine vitreous body with a strong Ca²⁺-mobilizing activity has been recently isolated to homogeneity by our group. In this work, a sequential analysis of its transmembrane signaling pathway has been undertaken to characterize the intracellular mechanisms responsible for the Ca²⁺ rise. The results show that this phospholipid induces, in a dose-dependent manner (ED₅₀ of around 0.25 µg/ml), a Ca²⁺ mobilization from inositol 1,4,5-trisphosphate-insensitive intracellular stores, with no participation of extracellular Ca²⁺. Upon repeated administration, it shows no signs of autologous desensitization, does not induce heterologous desensitization of the L--lysophosphatidic acid (LPA) receptor but is desensitized by the previous administration of LPA. The Ca²⁺-mobilizing activity requires a membrane protein, is blocked after preincubation of the cells with pertussis toxin and phorbol esters, as well as by U73122 (an inhibitor of phospholipases C/D), R59022 (a diacylglycerol kinase inhibitor), and D609 (which inhibits phosphatidylcholine-specific phospholipase C). Upon administration of this phospholipid, the intracellular levels of phosphatidic acid (PA) rise with a time course that parallels that of the Ca²⁺ mobilization, suggesting that PA could be responsible for this Ca²⁺ signal. Exposure to AACOCF₃ (a specific inhibitor of phospholipase A₂) does not modify the Ca²⁺ rise, ruling out the possibility that the PA generated could be further converted to LPA by the action of phospholipase A₂. Based on the experimental data obtained, a signaling pathway involving a phosphatidylcholine-specific phospholipase C coupled to diacylglycerol kinase is proposed. This compound may represent a new class of bioactive lipids with a putative role in the physiology of the vitreous body.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In recent years, the role of some bioactive lipids as molecules that participate actively in processes such as cell to cell communication, transmembrane signaling, and cellular activation, has been increasingly recognized. Since the discovery that L--lysophosphatidic acid (LPA)¹ acts as a growth-promoting signal molecule in many cellular systems (1), the list has grown to include sphingolipids (sphingosine, sphingosine 1-phosphate, sphingosylphosphoryl choline), ceramides, gangliosides, phospholipids (LPA, lysophosphatidyl serine), glycophospholipids, and even molecules as simple as free fatty acids (2-10). In this context, and as part of a research program of diabetic retinopathy (11), we have recently reported the isolation to homogeneity and preliminary chemical characterization of a new bioactive phospholipid from bovine vitreous body that acts as a potent Ca²⁺-mobilizing agent, provisionally called VLF (vitreous lipid factor) (12). Based on the requirements for activity, it has been concluded that this molecule is a high molecular weight phospholipid with two acyl groups in sn1 and sn2, and a complex polar head, including at least one terminal phosphate group and possibly a short peptide chain (12). This complex structure resembles that of glycosylphosphatidylinositols, which serve as anchors for some membrane proteins but have also been implicated in transmembrane signaling events (13).

In this work, the intracellular mechanism of action of this new and extremely potent activator of the Ca²⁺ pathway has been addressed. The delineation of its transmembrane signaling pathways can bring new light to our knowledge of the pathophysiology of the altered proliferation states of the eye.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- L--lysophosphatidic acid (oleoyl, LPA), L--phosphatidic acid (PA), L--phosphatidylcholine (PC), 1,2-dioleoyl-rac-glycerol (C18:1, [cis]-9; DG), phorbol 12-myristate 13-acetate (PMA), pertussis toxin (PTX), thapsigargin, and EGTA were obtained from Sigma. U73122, R59022,and D609 were obtained from Calbiochem. Fura-2 pentaacetoxymethylester (fura-2/AM) and the pentapotassium salt of fura-2 were obtained from Molecular Probes. Nickel(II) chloride was purchased from Merck.

Purification Procedure-- Lipid purification procedure has been described in detail elsewhere (12). Briefly, total lipids were extracted from bovine vitreous body homogenate with 4 volumes of chloroform:methanol (1:2) at room temperature. VLF was purified from crude lipids by solid phase extraction (Sep-Pack C₁₈), silica column chromatography, anion-exchange chromatography (DEAE-Sephadex, acetate form) and, finally, by high pressure liquid chromatography (cyanopropylsilyl-bonded silica). The activity was followed along the purification procedure by a [Ca²⁺]_i mobilization bioassay as described (12).

Cell Culture-- Although most of the cell lines tested were able to respond to this factor (11), all the experiments were carried out on the EGFR-T17 cell line, a clone of NIH3T3 (14). To measure the biological responses, cells were seeded in 100-mm dishes and cultured to 80% confluence for 2 days in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal calf serum. Media were supplemented with penicillin G (100 units/ml) and streptomycin sulfate (100 mg/ml). Cells were grown under a humidified atmosphere of 95% air, 5% CO₂ at 37 °C.

Calcium Measurements-- Intracellular [Ca²⁺]_i measurements were performed in cell suspensions using the fluorescent Ca²⁺ indicator fura-2. Cells were resuspended (2 × 100-mm plates/ml) in Krebs-Ringer-Hepes (KRH containing, in mM/liter, NaCl, 125; KCl, 5; MgSO₄, 1.2; KH₂PO₄, 1.2; CaCl₂, 2; glucose, 6; HEPES, 25, pH 7.4) and loaded with 3 µM fura-2/AM for 30 min at 37 °C under gentle continuous mixing. Cell suspensions were then diluted 1:4 with KRH and maintained at room temperature until use. For each measurement, around 2 × 10⁶ cells were resuspended in 2 ml of KRH and then placed in a cuvette positioned in a holder, thermostatically controlled at 37 ± 1 °C. The fluorescence signal was measured under continuous stirring in a LS-50B fluorimeter (Perkin-Elmer), adjusted to _ex = 345 and _em = 490 nm (15). Ca²⁺ readings were performed in ratio mode, using _1ex = 345, _2ex = 380, and _em = 490 nm, as described previously (4, 16) and calibrated by the cell lysis method (15). Extracellular Ca²⁺ measurements were performed with the cell-impermeant Ca²⁺ indicator fura-2 (0.25 µM) in Ca²⁺-efflux buffer (containing, in mM/liter, NaCl, 145; KCl, 5; glucose, 10; HEPES, 10, pH 7.4) essentially as described (17).

Inositol Phosphate Measurements-- Cells were labeled in 6-well multiwells by incubation in a 1:4 mixture of Dulbecco's modified Eagle's medium and inositol-free basal medium of Eagle, containing 10% dialyzed fetal calf serum (final inositol concentration, 10 µM), supplemented with myo-[2-³H]inositol (2 µCi/ml) for 48 h. The incubation medium was removed, and the monolayers were washed three times with KRH and then incubated for 10 min at 37 °C in KRH supplemented with 10 mM LiCl. Cells were stimulated with test agents for 30 min, after which the reactions were stopped, and the acid-soluble radioactivity extracted with 10% (v/v) trichloroacetic acid for 30 min on ice. After the centrifugation, the supernatants were washed with diethyl ether, and the total [³H]inositol phosphates were analyzed essentially as described (4, 18).

Phosphatidic Acid Measurements-- Cells were cultured in 6-well multiwells to a confluence of 80%, and then labeled with 1 µCi/ml of [³H]myristic acid in Dulbecco's modified Eagle's medium containing 0.25% fetal bovine serum. Before stimulation, cells were washed three times with KRH and incubated in this solution for 1 h. Cells were stimulated in this solution (freshly added) with the test agents at different times. Controls received an equivalent amount of vehicle (methanol). At the desired times, media were aspirated, and reactions immediately stopped by the addition of 2 ml of methanol to the plates. After 30 min, methanol extracts were transferred to glass tubes and mixed with 2 volumes of chloroform. To monitor the chromatographic separation, the following carrier lipids (30 µg each) were added to the samples: 1,2-sn-dioleoylglycerol, PA, and PC. Phase separation was achieved by the addition of 0.2 volume of water (19). The lipids present in the lower phase were separated on silica gel 60 TLC plates (Merck) using the upper phase of a mixture of ethyl acetate:isooctane:acetic acid:water (13:2:3:10 by volume) as the eluant (20, 21) (PA, R_f = 0.27; DG, R_f = 1.0; PC, R_f = 0.0). Lipid spots were visualized with iodine vapors, scraped off the plates, and prepared for liquid scintillation counting.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

When purified VLF was administered to EGFR-T17 cells, a prominent, transient [Ca²⁺]_i peak was observed that was followed by a small downward deflection of the fluorescence tracing and subsequently by a return to the prestimulatory levels (Fig. 1A). This Ca²⁺ peak was due exclusively to a redistribution from intracellular stores, as shown by the lack of effect of EGTA on the signal (Fig. 1B), the absence of a significant blockade in the presence of extracellular Ni²⁺ that blocks Ca²⁺ entry through Ca²⁺ channels (22) (Fig. 1C), and the absence of peak observed in cells pretreated for 30 min with 50 nM thapsigargin, a blocker of the sarco/endoplasmic reticulum Ca²⁺ pumps that empties the intracellular Ca²⁺ stores (23) (Fig. 1D). The slight downward deflection that follows the Ca²⁺ peak could in principle be the result of an efflux of Ca²⁺ to the extracellular medium. In fact, it was absent in the presence of extracellular Ni²⁺ or EGTA. However, the direct measurement of extracellular Ca²⁺ changes with fura-2 failed to detect any significant efflux after treatment of the cells with VLF, whereas a strong efflux was evident after the addition of LPA (Fig. 1E). The VLF-induced [Ca²⁺]_i rise was dose-dependent. As Fig. 1F shows, the response was half-maximal at around 0.25 µg/ml with saturation at doses in excess of 1 µg/ml. This would represent an ED₅₀ ranging from 250 nM for the simplest phospholipid molecule (phosphatidic acid) to less than 100 nM if we assume the presence of a phospholipid with a complex polar head including several sugar residues (12).

View larger version (16K): [in this window] [in a new window]   Fig. 1.   VLF induced calcium signal in EGFR-T17 cells. EGFR-T17 cells were stimulated with purified VLF, and changes in the intracellular Ca2+ were measured with the fluorescent probe fura-2. A, VLF-induced (1 µg/ml) calcium mobilization in control cells. B, VLF-induced calcium signal after elimination of the extracellular Ca2+ with excess EGTA (2.5 mM). C, inhibition of VLF calcium mobilization by Ni2+ ions (5 mM final concentration). D, effect of the sarco/endoplasmic reticulum Ca2+ pumps inhibitor thapsigargin (50 nM, 30 min). Under these conditions, Ca2+ is depleted from intracellular stores. E, effect of VLF on the Ca2+ efflux monitored with the cell-impermeant Ca2+-sensitive probe fura-2. Suspensions were prepared, and the effect of VLF (1 µg/ml) and LPA (1 µg/ml) on the rate of Ca2+ efflux assessed by fluorimetry (au, arbitrary fluorescence units). F, dose response of VLF-induced Ca2+ transients. The results (mean ± S.E. of three independent quadruplicate experiments) are expressed as the percentage of the maximum response.

The dose-response curve obtained for VLF (Fig. 1F) strongly suggests the presence of a saturable receptor molecule on the cell surface. To test this hypothesis, cell suspensions were treated with the cell-impermeant sulfhydryl reagent N-ethylmaleimide (NEM). This reagent oxydizes the cysteine residues at the extracellular domains of membrane proteins, rendering many of them inactive (24). As Fig. 2A shows, the preincubation (5 min) of the cells in the presence of 15 µM NEM resulted in a strong inhibition of the VLF-induced Ca²⁺ peak with no reduction of the Ca²⁺ peak elicited by ionomycin. The inhibition by NEM was dose-dependent, as shown in Fig. 2B, exceeding 75% at a dose of 15 µM. As a control, we used the Ca²⁺ ionophore ionomycin, a mobile ion carrier that acts by direct incorporation into the lipid bilayer and does not require any additional membrane protein for its function. No significant effect on the ionomycin-induced Ca²⁺ peak was evident at any of the NEM doses tested. It is interesting to note that after ionomycin treatment the post-stimulatory plateau is somewhat higher in NEM-treated cells than in control cells. This may reflect damage of the Ca²⁺ extrusion mechanisms at the plasma membrane after the treatment with NEM.

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Effect of N-ethylmaleimide on the VLF-induced Ca2+ transients. Cells were loaded with fura-2, and fluorescence expressed as the F345/F380 ratio. Ca2+ changes were induced by the addition of VLF (1 µg/ml) or the Ca2+ ionophore ionomycin (1 µM) either in control cells (A) or in cells pretreated for 5 min with the sulfhydryl reagent NEM (15 µM) (B). C, dose-response of the effect of NEM on the VLF- (1 µg/ml) or the ionomycin- (1 µg/ml) induced Ca2+ transients. Results are expressed as the percentage of the maximum response and correspond to a representative experiment repeated three times with comparable results.

In most cellular systems, the mobilization of Ca²⁺ from intracellular stores is usually triggered by activation of the phosphatidylinositol-specific phospholipase C (PI-PLC). This enzyme acts on phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂) at the internal leaflet of the plasma membrane generating inositol 1,4,5-trisphosphate (Ins(1,4,5)P₃), which will in turn trigger the release of Ca²⁺ from intracellular stores (25, 26). To test whether this mechanism could be responsible for the observed VLF-induced Ca²⁺ signal, the effect of U73122, a drug that has been extensively used as a specific inhibitor of PI-PLC (6) has been studied. As Fig. 3 shows, the preincubation of EGFR-T17 cells in the presence of U73122 (2.5 µM, 5 min) caused an almost complete blockade of the Ca²⁺ transients induced by VLF. U73122 exerts similar effects on the Ca²⁺ mobilization induced by LPA, a lipid that acts by activation of the PI-PLC (27, 28). In addition to Ins(1,4,5)P₃, the action of PI-PLC generates diacylglycerol (DAG), a second messenger that activates protein kinase C (PKC) (29). To assess the role, if any, of DAG-PKC on the VLF-activated signaling pathway, PKC activity was stimulated by means of the phorbol ester PMA. As Fig. 4 shows, the acute administration of PMA (1 µM, 5 min) caused a complete blockade of the VLF-induced Ca²⁺ transient (Fig. 4, A and B), a blockade that was prevented when the PKC activity was down-regulated by a chronic (1 µM, 24 h) exposure to the phorbol ester (Fig. 4C) (28).

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Effect of the PI-PLC inhibitor U73122 and pertussis toxin on the VLF-induced calcium signal. EGFR-T17 cells were stimulated with different doses of VLF in control cells () or in the presence of U73122 () (2.5 µM, 5 min) or PTX () (100 ng/ml, 2 h). Bar graph at right shows the effect of PTX (solid bar) or U73122 (dotted bar) on the Ca2+ signal induced by a saturating dose of LPA (1 µg/ml). Results (mean ± S.E. of three independent quadruplicate experiments) are expressed as the percentage of maximum Ca2+ response.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Effect of PMA on the VLF-induced calcium signal. Cells were stimulated with VLF (1 µg/ml) under control conditions (A) or after preincubation in the presence of the phorbol ester PMA (1 µM, 5 min) (B). PKC activity was down-regulated (C) by chronic (1 µM, 24 h) exposure to the phorbol ester prior to the new acute administration of PMA.

In the last few years it has been recognized that a simple phospholipid-derived molecule, LPA, is a potent chemical mediator that triggers different cellular responses in a number of cell lines (1, 30-32). To gain further insight into the mechanism of action of VLF, it was compared with LPA. Activation of intracellular Ca²⁺ mobilization is one of the earliest cellular signals elicited by LPA. As Fig. 5A shows, upon repeated administration, LPA induces homologous desensitization of its transmembrane signaling system and, in addition, induces heterologous desensitization to VLF. However, when the order of the stimuli was reversed (Fig. 5B), it was evident that VLF did not induce homologous desensitization of its receptor and was unable to cross-desensitize the LPA receptor. This result was surprising because if one assumes that the signaling is mediated by PI-PLC, DAG would be generated in parallel with Ins(1,4,5)P₃, triggering the activation of PKC, which in turn would close the system rendering it refractory to further activation by VLF or LPA.

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Cross-talk between the VLF and the LPA signaling pathways. Panel A, the repeated administration of a saturating dose of LPA (1 µg/ml) caused homologous desensitization as well as heterologous desensitization to VLF. Panel B, on the contrary, the repeated administration of a saturating dose of VLF (1 µg/ml) did not cause homologous desensitization nor heterologous desensitization to LPA.

To solve this discrepancy, the generation of inositol phosphates after stimulation of the cells with VLF and other stimuli, such as LPA, sphingosine 1-phosphate and epidermal growth factor, known to activate PtdIns(4,5)P₂ hydrolysis was studied. As Fig. 6 shows, VLF failed to induce formation of inositol phosphates from labeled PtdIns(4,5)P₂, a result that strongly questions the hypothesis that VLF induces Ca²⁺ mobilization via activation of PI-PLC. Furthermore, when the effect of PTX on the Ca²⁺ transient was evaluated, the preincubation of the cells with the toxin (100 ng/ml, 2 h) resulted in a virtually complete blockade of the VLF-induced Ca²⁺ signal (Fig. 3). In contrast, PTX caused only a slight reduction in the LPA-induced Ca²⁺ signal. The  subunits of the G-proteins that couple receptors to the phospholipase C-Ins(1,4,5)P₃-Ca²⁺ pathway belong to the G_q subfamily, and are not sensitive to PTX (33-35). For VLF, the G-protein involved appears to be a G_i, sensitive to PTX, and not coupled to PI-PLC. In fact, none of the products generated by this enzyme (Ins(1,4,5)P₃ and DAG), appear to rise after VLF stimulation of EGFR-T17 cells.

View larger version (13K): [in this window] [in a new window]   Fig. 6.   Effect of VLF, LPA, sphingosine 1-phosphate, and epidermal growth factor (EGF) on PtdIns(4,5)P2 hydrolysis. myo[2-3H]inositol-labeled EGFR-T17 cells were treated with VLF (5 µg/ml), LPA (5 µg/ml), sphingosine 1-phosphate (S1P) (5 µg/ml), or EGF (100 nM), and the levels of total [3H]inositol phosphates were measured. Data (mean ± S.E., cpm) are from three independent triplicate experiments.

In NIH 3T3 cells, the Ins(1,4,5)P₃-sensitive Ca²⁺ pool is dynamically coupled to extracellular Ca²⁺. When the Ca²⁺ levels in the Ins(1,4,5)P₃-sensitive Ca²⁺ pool are reduced immediately after the activation of the Ins(1,4,5)P₃-Ca²⁺ pathway by agonists that activate the hydrolysis of PtdIns(4,5)P₂ at the plasma membrane (LPA and EGF, for instance), a Ca²⁺ influx is triggered by aperture of capacitative Ca²⁺ channels (or store-operated channels) at the plasma membrane (36, 37). As the Ca²⁺ redistribution that follows the administration of VLF appears to originate from a Ca²⁺ pool not triggered by Ins(1,4,5)P₃, we analyzed the effect of VLF-induced Ca²⁺ redistribution on the Ca²⁺ influx. As Fig. 7A shows, the addition of thapsigargin to EGFR-T17 cells suspended in a nominally Ca²⁺-free medium was followed by a transient increase of the [Ca²⁺]_i due to the emptying of intracellular Ca²⁺ stores. When the extracellular Ca²⁺ was restored, a prominent capacitative Ca²⁺ influx was evident. A similar Ca²⁺ influx was evident after restoring the extracellular Ca²⁺ in cells prestimulated with LPA (Fig. 7B) or EGF (Fig. 7C) in Ca²⁺-free medium. On the contrary, when the cells were prestimulated with VLF in the absence of extracellular Ca²⁺, only a minor increase of Ca²⁺ was apparent after the restoration of the extracellular Ca²⁺ (Fig. 7D); however, this deflection of the fluorescence tracing was not different from the one observed when the extracellular Ca²⁺ was restored in control, nonstimulated cells (Fig. 7E). These results suggest that the Ca²⁺ pool mobilized by VLF is not functionally coupled to capacitative Ca²⁺ entry, a data that further reinforces the hypothesis that this pool is different from the Ins(1,4,5)P₃-sensitive Ca²⁺ pool.

View larger version (17K): [in this window] [in a new window]   Fig. 7.   Ca2+ influx in EGFR-T17 cells. Calcium measurements are performed in cells suspended in nominally Ca2+-free medium. At the end of the experiment, extracellular Ca2+ was restored (arrows) to evaluate Ca2+ influx. The addition of thapsigargin (50 nM) (A) induced a capacitative Ca2+ influx readily evident after restoration of the extracellular Ca2+. After the addition of LPA (1 µg/ml) (B), and EGF (10 nM) (C), the readdition of Ca2+ to the extracellular medium was also followed by a Ca2+ influx. The magnitude of the Ca2+ influx was very small in VLF-treated EGFR T17 cells (D) and not different from the one observed in control, nonstimulated cells (E).

Ca²⁺ mobilization from non-Ins(1,4,5)P₃-sensitive Ca²⁺ stores is activated in some cellular systems by phosphatidylcholine-specific phospholipase C (PC-PLC), an enzyme that hydrolyzes phosphatidylcholine generating diacylglycerol and phosphocholine (38, 39). The newly formed DAG is then converted by the action of a diacylglycerol kinase (DAG-K) to PA, which in turn will activate Ca²⁺ redistribution from internal stores (40-42). To check the possibility that VLF could be using this signaling pathway, the DAG-K inhibitor R59022 (42-44) was used. As Fig. 8, A and B shows, the preincubation (10 µM, 30 min) in the presence of this inhibitor caused a clear-cut blockade of the Ca²⁺ peak, strongly suggesting that this pathway could be taking part in the triggering of the Ca²⁺ redistribution. In addition, the reduction of the Ca²⁺ transients observed after preincubating the cells with the PC-PLC inhibitor D609 (39, 45) (10 µM, 5 min) (Fig. 8C) reinforced this interpretation. The inhibitory effects of both R59022 and D609 appear to be specific, as no inhibition was apparent when the cells were stimulated with either EGF or LPA (Fig. 8E). Furthermore, the lack of effect of the PLA₂-specific inhibitor AACOCF₃ (10 µM, 5 min) on the Ca²⁺ rise (Fig. 8D), strongly supports the hypothesis of a direct action of PA on the intracellular Ca²⁺ stores, rather than an indirect one mediated by its conversion to LPA by PLA₂, as has been demonstrated for sphingosylphosphorylcholine (46). In fact, when the levels of intracellular Ca²⁺ and PA were correlated in VLF- or LPA-stimulated cells (Fig. 9, A and C), it became clear that the administration of purified VLF, but not LPA, was followed by a rapid and transient increase of the PA levels (Fig. 9B), and the time course of the PA rise in VLF-stimulated cells closely matched the time course of the Ca²⁺ elevation, strongly suggesting that the PA rise may be in fact the biochemical signal triggering the Ca²⁺ transient in VLF-stimulated cells.

View larger version (15K): [in this window] [in a new window]   Fig. 8.   Effect of DAG-K, PC-PLC, and PLA2 inhibitors on the VLF-induced Ca2+ mobilization. A, control cells stimulated with a saturating dose (1 µg/ml) of purified VLF. B, cells pretreated with the DAG-K inhibitor R59022 (10 µM, 30 min) before the administration of VLF. As the inhibition of DAG-K causes accumulation of DAG, the experiment was performed in the presence of the PKC inhibitor staurosporine (1 µM) to avoid interference. C, cells pretreated with the PC-PLC inhibitor D609 (10 µM, 5 min) and then stimulated with a saturating dose (1 µg/ml) of purified VLF. D, cells pretreated with the PLA2 inhibitor AACOCF3 (10 µM, 5 min) before the VLF stimulation. E, dose response of the effect of R59022 and D609 on the Ca2+ rise induced by saturating doses of VLF (1 µg/ml), LPA (1 µg/ml), and EGF (10 nM). Neither of the inhibitors affected the Ca2+ responses elicited by LPA and EGF. The results (mean ± S.E.) are expressed as the percentage of the maximum response.

View larger version (14K): [in this window] [in a new window]   Fig. 9.   Time courses of the Ca2+ and PA rises in cells stimulated with VLF and LPA. Parallel subconfluent dishes were: (i) resuspended and loaded with fura-2 for high speed monitorization of the Ca2+ levels in response to VLF (1 µg/ml, arrow) (A), or LPA (1 µg/ml) (C); (ii) labeled with [3H]myristic acid for the determination of PA generation after stimulation with VLF or LPA (both at 5 µg/ml) by TLC (B). Results (mean ± S.E.) are from three independent determinations and expressed in B as the increase (in cpm) over control values.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

From the analysis of the data presented so far, the signal transduction pathway used by VLF, the complex lipid isolated by our group from bovine vitreous body to induce Ca²⁺ mobilization, can be delineated. This Ca²⁺ signal is generated exclusively by redistribution from internal stores, with no significant participation of the extracellular Ca²⁺. It requires the presence of a membrane protein, the inactivation of which by NEM greatly reduces the ability of VLF to induce Ca²⁺ mobilization, and determines a dose-response curve showing half-maximal saturation in the nanomolar range. The coupling between this putative membrane receptor and the intracellular effectors is mediated by a PTX-sensitive G-protein, as shown by the virtually complete blockade of the Ca²⁺ signal after PTX treatment of the cell suspensions. In a similar way, the phospholipase C (PLC) blocker U73122 was able to suppress the Ca²⁺ mobilization response. The system appears to be under regulation by PKC, because phorbol esters cause a complete suppression of the response, which disappears when the endogenous PKC activity is down-regulated by chronic exposure to the phorbol ester. Taken together, the data obtained resembled the transmembrane signaling pathway described for LPA (27). However, a closer analysis revealed some differences. First, upon repeated administration, LPA causes an autologous desensitization that renders the cell unresponsive to further stimulation with this lipid. After the repeated administration of VLF, on the contrary, no signs of autologous desensitization are evident, despite the clear-cut heterologous desensitization that LPA induces on the VLF Ca²⁺ signal. Second, the Ca²⁺ pool mobilized by LPA is Ins(1,4,5)P₃-sensitive, and consequently the administration of LPA is followed by a stimulation of Ins(1,4,5)P₃ formation. No such stimulation was evident, however, after the administration of VLF. Furthermore, for receptors coupled to PI-PLC by G-proteins, the G subtype involved is a G_q, that is not sensitive to PTX (47), as it happens for LPA. For VLF, however, both the exquisite sensitivity to PTX and the lack of detectable Ins(1,4,5)P₃ generation exclude a G_q as the mediator between the receptor and the effector. This is in contrast with the data obtained after exposure of the cells to U73122, an inhibitor of PI-PLC. However, several reports have pointed out that the action of this drug is far from being specific, being able to also inhibit other PLCs, as well as phospholipase D (48, 49). Taking this into account, the Ca²⁺ mobilization induced by VLF could be explained by two alternate routes; either the PTX-sensitive G-protein is coupled to phospholipase D, inducing the intracellular accumulation of PA, or coupled to a different PLC, such as PC-PLC. The sensitivity to PKC activation strongly suggests that the latter possibility is more plausible, as the activation of PKC is followed in most systems by activation of phospholipase D activity and/or blockade of PC-PLC (29, 50, 51). Furthermore, the preincubation of the cells with inhibitors of both PC-PLC and DAG-K caused a complete blockade of the VLF-induced Ca²⁺ signal, whereas the preincubation with the PLA₂ inhibitor AACOCF₃ was devoid of action (Fig. 8). These results strongly suggest that the Ca²⁺ signal was in fact due to a direct rise in the intracellular PA levels and not to an indirect action mediated by a PLA₂-mediated conversion of PA to LPA, as has been demonstrated for other systems (46). In fact, the measurement of the time course of PA generation in VLF-stimulated cells unambiguously shows that the PA levels rise in response to the VLF administration with a time course comparable to the Ca²⁺ mobilization (Fig. 9). It thus appears that, in our experimental system, PA is in fact the signal that triggers the Ca²⁺ rise from Ins(1,4,5)P₃-independent stores rather than a mere consequence of the Ca²⁺ elevation, as it has been suggested for other systems (21).

Taken together, the data obtained in the present study strongly suggest that VLF acts through a signaling pathway different to the one used by LPA and other bioactive lipids (Fig. 10). Upon ligand binding, the receptor activates a PC-PLC through a PTX-sensitive G-protein. The activation of PC-PLC results in the generation of phosphocholine and DAG. The latter would be rapidly converted to PA by means of a DAG-K associated with the receptor-G-protein complex, with virtually no activation of PKC, so the system would not show, as observed, homologous or heterologous desensitization of the LPA transmembrane signaling system (Fig. 5). In fact, it has been recently shown that DAG-K is topologically restricted in the plasma membrane and physically coupled to activated receptors (41). The PA, in turn, would act on intracellular, non-Ins(1,4,5)P₃-sensitive stores, triggering the observed Ca²⁺ transients.

View larger version (23K): [in this window] [in a new window]   Fig. 10.   Proposed model for the signal transduction pathway used by VLF to trigger intracellular Ca2+ mobilization. The administration of VLF to cells activates a PC-PLC that hydrolyzes phosphatidylcholine at the plasma membrane. The DAG generated is converted to PA by means of a DAG-K coupled to the system. PA then releases Ca2+ from intracellular Ins(1,4,5)P3-insensitive Ca2+ pools. No activation of PKC by DAG occurs. On the contrary, the addition of LPA activates a PI-PLC, which generates Ins(1,4,5)P3 and DAG from phosphatidylinositol 4,5-bisphosphate (PIP2), with liberation of Ca2+ from Ins(1,4,5)P3-sensitive stores and activation of PKC. Dotted lines indicate negative regulation. LPA-R, LPA receptor; MAP, mitogen-activated protein.

The biological role of this new class of bioactive lipids in the physiology and pathophysiology of the vitreous body has not been ascertained as of yet. The identification of this new class of lipids, with strong cellular activation properties could have important and unexpected implications in our understanding of the pathophysiology of altered proliferation at the vitreous body/retina.

	ACKNOWLEDGEMENTS

The expert technical assistance of Mary Lage is greatly acknowledged.

	FOOTNOTES

^* This work was supported by a research grant from Fondo de Investigación Sanitaria, Spanish Ministry of Health.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. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Medicine, Cellular Endocrinology Laboratory, Compostela University School of Medicine, P. O. Box 563, E-15780 Santiago de Compostela, Spain. Tel. and Fax: 34-981-57-21-21; E-mail: meffcasa@usc.es.

	ABBREVIATIONS

The abbreviations used are: LPA, L--lysophosphatidic acid; VLF, bovine vitreous lipid factor; PA, L--phosphatidic acid; PC, L--phosphatidylcholine; PTX, pertussis toxin; NEM, N-ethylmaleimide; PLC, phospholipase C; PI-PLC, phosphatidylinositol-specific PLC; PC-PLC, phosphatidylcholine-specific PLC; DAG, diacylglycerol; DAG-K, DAG kinase; PLA₂, phospholipase A₂; PKC, protein kinase C; PtdIns(4,5)P₂, phosphatidylinositol 4,5-biphosphate; Ins(1,4,5)P₃, inositol 1,4,5-trisphosphate; EGF, epidermal growth factor; EGFR, EGF receptor; KRH, Krebs-Ringer-Hepes.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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