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(Circulation Research. 1995;77:888.)
© 1995 American Heart Association, Inc.

Articles

Effects of Lysophosphatidic Acid, a Novel Lipid Mediator, on Cytosolic Ca²⁺ and Contractility in Cultured Rat Mesangial Cells

Chiyoko N. Inoue, Hayley G. Forster, Murray Epstein

From the Nephrology Section, Miami (Fla) VA Medical Center and The University of Miami School of Medicine.

Correspondence to Murray Epstein, MD, Nephrology Section (111C1), Miami VA Medical Center, 1201 NW 16th St, Miami, FL 33125.

	Abstract

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Abstract Lysophosphatidic acid (LPA), the smallest and structurally simplest phospholipid, has the potential to modulate cellular signaling in diverse tissues and cell types, including fibroblasts. In the present study, we assessed the effects of LPA on cultured rat glomerular mesangial cells. Quantitative changes of [Ca²⁺]_i in response to LPA were measured in monolayers of mesangial cells loaded with the fluorescent Ca²⁺ indicator fura 2. LPA (10 nmol/L to 100 µmol/L) increased [Ca²⁺]_i in a dose-dependent manner and evoked inositol trisphosphate formation. LPA (1 µmol/L to 30 µmol/L) also elicited a marked, albeit transient, contractile response in mesangial cells cultured on collagen gel, as assessed by a decrease in cell surface area (CSA). The contraction persisted for 5 minutes (CSA decreased by 31%), whereupon the mesangial cells gradually relaxed with a consequent increase in CSA. Pretreatment of mesangial cells with isradipine (1 µmol/L), a dihydropyridine-sensitive Ca²⁺ channel blocker, completely blocked LPA-induced contraction. Isradipine also decreased resting [Ca²⁺]_i levels as well as the peak and the subsequently sustained [Ca²⁺]_i levels induced by LPA, suggesting that the contractile effects of LPA are dependent on Ca²⁺ entry through voltage-gated Ca²⁺ channels. Finally, LPA stimulated an increase in both prostaglandin E₂ synthesis and cAMP accumulation, indicating that these mediators may modulate the contractile effects of LPA. Our study is the first demonstration that exogenous LPA induces mesangial cell contraction and suggests that the contraction is mediated by mobilization of intracellular Ca²⁺ by activation of the phosphoinositide cascade as well as by promotion of Ca²⁺ entry across the plasma membrane.

Key Words: lysophosphatidic acid • mesangial cells • phosphoinositide cascade • Ca²⁺ signaling • Ca²⁺ antagonists

	Introduction

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The glomerular mesangium plays an important role in renal physiology. The ability of the mesangium to contract and relax in response to a number of vasoactive substances, cytokines, and growth factors allows it to regulate renal blood flow and glomerular filtration rate.¹ In addition, because of the proximity of mesangial cells to the glomerular microcirculation, many circulating and locally produced cellular products that have been implicated in glomerular injury can directly influence mesangial function.^{2 3}

Recently, increasing evidence has accumulated delineating an important role for platelets and platelet metabolites, including PAF, PDGF, and thrombin, in mediating inflammatory and thrombotic kidney disorders.^{4 5} The activation of platelets and the release of their metabolites into the extracellular space induce profound responses in the glomerular mesangium, including contraction, proliferation, and cytokine production.^{6 7}

LPA, a structurally simple phospholipid, has currently attracted increasing investigative interest as a platelet metabolite capable of inducing significant physiological effects.^{8 9} Schumacher et al¹⁰ have demonstrated that LPA stimulates platelet aggregation. Furthermore, LPA is rapidly produced in the plasma membrane of thrombin-activated platelets and growth factor–stimulated fibroblasts.¹¹ The production of LPA in activated cells through activation of PLA₂ and PLD suggests a possible role for LPA as a second messenger.¹² Recently, Wada et al¹³ have demonstrated that LPA is produced by mesangial cells when stimulated by exogenous group II PLA₂. Because PLA₂ is considered to play a role in the initiation and propagation of inflammatory reactions in mesangial cells,¹⁴ it is reasonable to speculate that PLA₂ may exert its effects in part through LPA in the glomerular microcirculation.

Collectively, these diverse properties of LPA provide a theoretical framework for considering a possible pathophysiological role for LPA in the glomerular mesangium. Consequently, we conducted the present studies to characterize the effects of LPA on cultured rat mesangial cells. In the present study, we provide the first evidence demonstrating that exogenous LPA leads to the activation of phosphoinositide hydrolysis and intracellular Ca²⁺ mobilization, consequently evoking a marked contraction of cultured mesangial cells.

	Materials and Methods

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Materials
RPMI 1640, FCS, and fatty acid–poor fraction V BSA were obtained from GIBCO. Rat tail collagen type I was purchased from Collaborative Biomedical Products. Fura 2-AM was obtained from Molecular Probes. L--LPA (1-oleoyl) and all other biochemicals were obtained from Sigma Chemical Co. Isradipine, a DHP-sensitive Ca²⁺ antagonist, was kindly provided by Sandoz Pharmaceuticals Corp.

Primary Culture of Rat Mesangial Cells
Isolated glomeruli were prepared from the kidney cortices of five male Sprague-Dawley rats weighing 75 to 100 g by consecutive sieving with three different stainless steel meshes (106, 150, and 75 µm), as reported previously by Kreisberg and Karnovsky.¹⁵ The glomeruli were then digested with collagenase and seeded onto plastic culture flasks. Cells were maintained in culture in RPMI 1640 medium supplemented with 20% FCS, ITS premix, penicillin (50 U/mL), and streptomycin (50 µg/mL) at 37°C in 5% CO₂ atmosphere. Mesangial cell growth predominated by day 14 of the primary culture, and the first subculture was performed on day 21. Cells in the present study were used in the third to seventh passages because they satisfied the previously reported criteria for mesangial cells.¹⁵

Contraction Studies
The culture of the rat mesangial cells for the contraction studies was performed in accordance with well-established and characterized techniques as described by Simonson and Dunn.¹⁶ Briefly, 7 vol of type I collagen gel solution was mixed with 2 vol of 5x concentrated DMEM and 1 vol of HEPES/NaOH buffer. Aliquots (1 mL) of the mixture were plated into culture dishes (35 mm diameter) and allowed to gel by warming to 37°C for 30 minutes. Rat mesangial cells from passages 3 to 7 were then subcultured onto the type I collagen gel bed. Contraction was tested 24 hours after subculture onto the collagen gel by incubating cells with or without 30 µmol/L LPA in DMEM containing 25 mmol/L HEPES (pH 7.4) and 0.1% BSA at 37°C. Shape changes of the cells were followed sequentially by using a phase-contrast microscope (Nikon Diaphot-TMD) coupled to a high-resolution video monitor (Triniton, model PMV-1343MD, Sony Corp) and a color video printer (Mavigraph, model UP-3000, Sony Corp). Black and white photomicrographs were taken at intervals of 3 to 5 minutes from images captured on the monitor via a CCD video camera system (model VI-470, Optronics Engineering) and digitally enhanced with an Omnex/PM video processor (Imagen Instrumentation Inc). Photomicrographs of the cells were then manually traced, and the tracings were scanned with a Hewlett-Packard Scanjet IIc. The scanned images were saved in an eight-bit TIFF format at 75 dpi. Object surface area (ie, CSA) was measured by using the automated flood-fill method in SIGMA SCAN/IMAGE 1.2 for WINDOWS (Jandel Scientific). Cells were scored positively for contraction when the decrease of CSA was >7%.

Ca²⁺ Measurements
Mesangial cells from passages 4 to 7 were subcultured onto plastic Aclar coverslips (Allied Engineered Plastics) and grown to confluence. At this point, the cells were serum-deprived by decreasing the media concentration (RPMI 1640) of FCS to 0.5% for 2 days. Before the fluorescence studies were conducted, the cells were loaded with 3 µmol/L fura-2-AM for 30 minutes at 37°C in RPMI, washed three times, and then incubated again for 20 minutes in fura 2-free RPMI to allow for intracellular dye cleavage. The coverslips were inserted diagonally into quartz cuvettes containing 2.5 mL of KHH buffer (mmol/L: NaCl 140, KCl 5, MgSO₄ 1, Na₂HPO₄ 1, CaCl₂ 1, glucose 25, and HEPES 25, pH 7.2) supplemented with 0.05% BSA and maintained at 37°C with constant stirring. [Ca²⁺]_i was determined by measuring the fluorescence of Ca²⁺/fura 2 with a spectrofluorometer (model RF-M2001, Photon Technology International). Excitation wavelengths were set at 340 and 380 nm, and emission was monitored at 510 nm. Maximal fluorescence was determined with 5 µmol/L ionomycin, followed by minimal fluorescence with 5 mmol/L EGTA. The values for [Ca²⁺]_i were calculated according to the method of Grynkiewicz et al¹⁷ as follows: [Ca²⁺]_i=K_d[(R-R_min)/(R_max-R)]x(380_min/380_max), where R_min is the fluorescence ratio (R) of 340/380 when EGTA was added, and R_max is the ratio when ionomycin was added; K_d for fura 2 was 224 nmol/L.¹⁷

Measurement of IP₃ Production
Confluent cells in 35-mm-diameter dishes were preincubated for 3 hours in serum-free RPMI containing 0.1% BSA and then subsequently for 10 minutes in DMEM containing 25 mmol/L HEPES (pH 7.4), 0.1% BSA, and 10 mmol/L LiCl. Stimulation was initiated by adding LPA to a final concentration of 30 µmol/L, and the reaction was terminated at 30 and 60 seconds by the rapid removal of the medium and the addition of ice-cold 15% trichloroacetic acid to the dishes. After being placed on ice for 30 minutes, the cells were scraped off and sedimented by centrifugation. The acid extract (supernatant) was neutralized by washing five times with 8 vol of water-saturated diethyl ether. The sample was dried with a vacuum concentrator (Savant Instruments Inc) and dissolved in distilled water. IP₃ was assayed with an IP₃-specific binding assay kit from Amersham Corp.

[³H]Arachidonate Release and PGE₂ Synthesis Assay
For measuring [³H]arachidonate release, subconfluent monolayers of cells in six-well plates were prepared. Cells were incubated for 20 hours with 1 µCi/mL [³H]arachidonate in culture medium, washed three times with serum-free RPMI containing 0.1% BSA, and subsequently incubated for 3 hours in RPMI containing 0.1% BSA and then for 30 minutes in DMEM buffer containing 25 mmol/L HEPES (pH 7.4) and 0.1% BSA. After the final wash, 1.5 mL of fresh DMEM buffer with or without 30 µmol/L LPA was added to the plates. Aliquots of media were removed without replacement at predetermined intervals (3, 5, 10, 20, and 30 minutes) and processed for liquid scintillation. Counts were corrected for volume.

For measurements of PGE₂ synthesis, subconfluent cells in six-well culture dishes were prepared. After 3 hours of incubation in serum-free RPMI containing 0.1% BSA, the medium was changed to DMEM buffer containing 25 mmol/L HEPES (pH 7.4) and 0.1% BSA for 30 minutes. At the onset of the experiment, 1 mL of fresh DMEM buffer with or without 30 µmol/L LPA was added to the wells, followed by incubation for 5, 10, and 20 minutes. The supernatants were immediately collected at the indicated intervals, placed on dry ice, and kept frozen at -80°C until assay. Immunoreactive PGE₂ was measured by radioimmunoassay using a [¹²⁵I]PGE₂ assay kit from Amersham.

Measurements of Intracellular cAMP Accumulation
Confluent monolayers of cells in six-well culture dishes were preincubated for 3 hours in serum-free RPMI containing 0.1% BSA and subsequently for 30 minutes in DMEM containing 25 mmol/L HEPES (pH 7.4) and 0.1% BSA. At time 0, the medium was aspirated and replaced with medium containing 0.2 mmol/L IMX or medium containing 0.2 mmol/L IMX plus 30 µmol/L LPA. After incubation for the indicated times (3, 5, 10, and 20 minutes), the supernatants were discarded, and the cyclic nucleotides were extracted by adding ethanol containing 20 mmol/L HCl to the cells for 30 minutes on ice. Cells were then scraped from the dishes and transferred to microcentrifuge tubes, and cell suspensions were sonicated. The suspension was then centrifuged (12 000g) for 10 minutes at 4°C to precipitate the protein, and the supernatant was collected. The supernatant was vacuum-dried and dissolved in an adequate volume of 50 mmol/L Tris/4 mmol/L EDTA (pH 7.5). cAMP content was determined by using the [³H] cAMP assay kit from Amersham. Protein determinations were performed by using the Bio-Rad kit, with BSA used as a standard.

Statistics
Data were expressed as mean±SEM. Data were analyzed by one-way ANOVA followed by the unpaired t test. For the comparison of responding versus nonresponding cells in the contraction studies using LPA, ² analysis was used. A value of P<.05 was considered statistically significant.

	Results

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LPA-Induced Contraction of Mesangial Cells
Contraction studies were performed by using rat mesangial cells cultured on three-dimensional collagen gel. As shown in Table 1, a progressively larger number of mesangial cells contracted in response to increasing concentrations of LPA. Maximal contraction was observed at a concentration of 30 µmol/L of LPA, where 59% of the mesangial cells responded with a decrease in CSA. In contrast, only 8% of the control cells (ie, HEPES-buffered DMEM alone) manifested significant reduction in CSA.

View this table: [in this window] [in a new window]   Table 1. Reduction of CSA in Response to LPA in Mesangial Cells

The shape changes induced by LPA in rat mesangial cells cultured on type I collagen gel by LPA were photographed over time (Fig 1), and the magnitude of contraction was summarized in Fig 2. The onset of contraction was detected as early as 1 minute after the addition of LPA. Contraction progressed for 3 to 5 minutes, at which time CSA was minimal, and the mean reduction of CSA was -31±2.8% of the control value. However, in the continuous presence of LPA, almost all of the contracted cells gradually relaxed. By 30 minutes, CSA had exceeded the control (time 0) values by +30%. A second application of LPA (30 µmol/L) to the same responsive cells failed to alter their shape. Thus, LPA-induced shape changes are subject to homologous desensitization, as one would anticipate for a receptor-mediated process.

View larger version (74K): [in this window] [in a new window]   Figure 1. Phase-contrast microscopy of cultured mesangial cells in the presence of LPA. A, Cells in DMEM containing 25 mmol/L HEPES (pH 7.4) and 0.1% BSA. B, Five minutes after incubation with 30 µmol/L LPA. LPA stimulates contraction of rat glomerular mesangial cells. Arrows note diminution of CSA and the shortening of cytoplasmic extensions. C, Thirty minutes after exposure to LPA. Arrows indicate that contracted cells relaxed and CSA increased. Magnification x600.

View larger version (14K): [in this window] [in a new window]   Figure 2. Time course of contractile response of rat mesangial cells that responded to LPA. At time 0, monolayers were stimulated with 30 µmol/L LPA. After 5 minutes, the contracted mesangial cells manifested a spontaneous attenuation of the contracted state and relaxed progressively. By 30 minutes, CSA exceeded the control (time 0) values. Results are mean±SEM (n=16).

We next characterized the determinants of contractility by assessing the effects of LPA on mesangial cells in the presence of a Ca²⁺ channel blocker or 2 mmol/L EGTA (Table 1). Contraction in response to LPA was completely blocked by pretreatment for 3 minutes with 1 µmol/L isradipine, a DHP-sensitive Ca²⁺ channel blocker.¹⁸ This was the minimal effective dose of isradipine required to block contraction; eg, 0.5 and 0.1 µmol/L isradipine failed to inhibit LPA-induced contraction. In addition, chelation of extracellular Ca²⁺ with EGTA pretreatment for 3 minutes completely inhibited LPA-induced contraction (Table 1). These data indicate that Ca²⁺ entry across the plasma membrane is essential in mediating LPA-induced contraction of mesangial cells.

We then characterized the effects of cyclooxygenase inhibition on LPA-induced mesangial cell contractility. Pretreatment of the cells for 3 minutes with indomethacin (10 µmol/L) induced a mild but significant decrease in basal CSA (-6.6±2.5%, P<.05). The addition of LPA (30 µmol/L) elicited a further constriction in 15 (71%) of the 21 cells that progressed for the duration of the experiment (CSA, -24±2.7% at 5 minutes and -34±3.9% at 30 minutes). These data contrast with our findings in cells not treated with indomethacin, in which the contractile response persisted rather than abated over time.

We next examined the effect of 30 µmol/L lysophosphatidyl choline, the structural analogue of LPA, for the ability to induce contraction in mesangial cells (Table 1). In contrast to LPA, this analogue failed to produce significant contraction during a 30-minute incubation on collagen gel at 37°C. Furthermore, pretreatment of mesangial cells with cytochalasin B (0.5 mg/mL), an inhibitor of actin polymerization, completely blocked LPA-induced cell contraction and cell retraction (Table 1). In contrast, colchicine, a microtubule-disrupting drug, did not inhibit LPA-induced contraction of mesangial cells (data not shown). In concert, these observations indicate that for LPA-induced contraction, DHP-sensitive Ca²⁺ channels are essential and the contractile response is mediated by actin-based contractile forces.

Effects of LPA on Cytosolic Ca²⁺
Because increases of [Ca²⁺]_i by agonists constitute the principal component for mesangial cell contraction, we examined the effects of LPA on [Ca²⁺]_i in rat mesangial cells by using confluent monolayers of cells loaded with the fluorescent Ca²⁺ indicator fura 2. The average resting [Ca²⁺]_i levels in rat mesangial cells in KHH buffer were estimated at 105±4.4 nmol/L (n=17) (Table 2), a value that is in good agreement with those previously published for these cells.¹⁹ The addition of LPA to the bathing solution induced a rapid and transient elevation in [Ca²⁺]_i, which peaked within 15 to 30 seconds and then declined to a new steady level that was higher than resting levels (Fig 3A). Peak [Ca²⁺]_i responses increased in a dose-dependent manner with a maximal intracellular Ca²⁺ elevation at 100 µmol/L (575±47.2 nmol/L, n=6) (Fig 4). LPA concentrations of >100 µmol/L did not induce any further increases in peak [Ca²⁺]_i, and measurable Ca²⁺ transients were observed for LPA concentrations as low as 10 nmol/L.

View this table: [in this window] [in a new window]   Table 2. Effects of Differing Extracellular Conditions on [Ca2+]i Levels in Mesangial Cells

View larger version (14K): [in this window] [in a new window]   Figure 3. Representative tracings depicting changes in Ca2+/fura 2 fluorescence induced by LPA in cultured rat mesangial cells during differing extracellular conditions. Confluent cell monolayers loaded with fura 2 were preincubated in KHH buffer containing 1 mmol/L Ca2+ (A), Ca2+-free KHH buffer containing 2 mmol/L EGTA (B), and KHH buffer containing 1 µmol/L isradipine (C). Calculated values for [Ca2+]i are shown on the ordinates.

View larger version (12K): [in this window] [in a new window]   Figure 4. LPA concentration-response curve of peak Ca2+/fura 2 signals. Fura 2–loaded cells were incubated with LPA (1 nmol/L to 100 µmol/L), and the agonist-induced increment of [Ca2+]i was calculated from basal levels. Each point is the mean±SEM of four determinations. *P<.05 vs unstimulated control cells.

To determine whether the increase in intracellular Ca²⁺ induced by LPA was due to Ca²⁺ release from intracellular stores, experiments were repeated with the cells in a Ca²⁺-free KHH buffer in the presence of 2 mmol/L EGTA. The absence of extracellular Ca²⁺ slightly decreased the resting levels of [Ca²⁺]_i (90±6.5 nmol/L, n=7, P>.05) but significantly reduced the LPA-induced increase in intracellular Ca²⁺ transients (428±23.0 nmol/L, n=7, P<.05) (Fig 3B, Table 2). On the other hand, pretreatment with the Ca²⁺ channel blocker isradipine in a KHH buffer containing 1 mmol/L of Ca²⁺ not only significantly decreased resting [Ca²⁺]_i levels to 59±3.1 nmol/L (n=7, P<.01) but also markedly reduced the peak value of [Ca²⁺]_i as well (259±35.6 nmol/L, n=7, P<.01) (Fig 3C, Table 2). These results suggest that whereas LPA, in common with many Ca²⁺-mobilizing agents, acts primarily by releasing Ca²⁺ from internal stores, it also acts in part by promoting Ca²⁺ entry across the plasma membrane. Furthermore, it should be noted that even after an LPA-induced intracellular Ca²⁺ transient stabilized to a new resting steady state level of [Ca²⁺]_i, reexposure of the mesangial cells to LPA did not elicit a significant response. However, these monolayers were still able to respond to ET-1 (100 nmol/L) with an increase in [Ca²⁺]_i similar to that elicited by ET-1 alone, albeit to a lesser extent (Fig 5). In other words, the LPA-induced Ca²⁺ mobilization response is subject to homologous, but not heterologous, desensitization.

View larger version (13K): [in this window] [in a new window]   Figure 5. LPA-stimulated mesangial cells loaded with fura 2 displayed homologous desensitization upon reexposure to LPA. Preincubation with 100 µmol/L LPA for 3 minutes diminished the subsequent LPA-induced intracellular Ca2+ transient but had no effect on the ET-1 (100 nmol/L)–induced intracellular Ca2+ transient.

IP₃ Formation
Phosphoinositide hydrolysis by PLC is thought to constitute a crucial component of the constrictive signal of many vasoconstrictors.²⁰ Therefore, we examined the ability of LPA to stimulate phosphoinositide metabolism in the presence of 10 mmol/L LiCl. When cultured rat mesangial cells were stimulated with 100 µmol/L LPA, IP₃ levels rapidly increased from their basal values to reach peak values at 30 seconds and then subsequently returned to basal values by 60 seconds (Fig 6). The peak IP₃ values 30 seconds after stimulation were 1.6-fold greater than the basal values. These results indicate that LPA stimulates the hydrolysis of phosphatidylinositol 4,5-diphosphate by PLC and suggest that the mobilization of Ca²⁺ from intracellular Ca²⁺ stores is mediated by IP₃.

View larger version (12K): [in this window] [in a new window]   Figure 6. Effects of LPA on IP3 formation in cultured rat mesangial cells as a function of time. Confluent cells were incubated with LPA (100 µmol/L) in DMEM containing 25 mmol/L HEPES (pH 7.4), 0.1% BSA, and 10 mmol/L LiCl for the indicated times. Each point is the mean±SEM of quadruplicate dishes. *P<.05 vs value at time 0.

[³H]Arachidonate Release and PGE₂ Synthesis
To test whether LPA stimulates the release of arachidonic acid from membrane phospholipids into the medium, we measured the release of [³H]arachidonate and the synthesis of PGE₂, the dominant arachidonate metabolite in rat mesangial cells.²¹ When added to mesangial cells prelabeled with [³H]arachidonate, LPA (30 µmol/L) evoked a time-dependent increase in [³H]arachidonate release that plateaued by 20 minutes (Fig 7). LPA (30 µmol/L) also increased the production of immunoassayable PGE₂. As shown in Fig 8, LPA stimulated a threefold increase in PGE₂ synthesis by 5 minutes (basal value, 110±20 pg/mL; 5-minute value, 330±60 pg/mL; P<.01), which remained constant for at least 20 minutes. In the control mesangial cells, PGE₂ production was not stimulated. These results indicate that the exposure of mesangial cells to LPA stimulated arachidonic acid release and PGE₂ synthesis in rat mesangial cells.

View larger version (16K): [in this window] [in a new window]   Figure 7. Release of [3H]arachidonate in response to LPA. Rat mesangial cells prelabeled with [3H]arachidonate were stimulated with 30 µmol/L LPA (), and the radioactivity released into the medium was measured at the times indicated. Each point is the mean±SEM of quadruplicate dishes. *P<.05 vs unstimulated control cells (•).

View larger version (13K): [in this window] [in a new window]   Figure 8. Effects of LPA on PGE2 production as a function of time in cultured rat mesangial cells. After the addition of 30 µmol/L LPA (), cells were incubated for the indicated times, and PGE2 generation in the culture medium was determined. Each point is the mean±SEM of triplicate dishes. *P<.05 vs unstimulated control cells (•).

cAMP Accumulation
To further elucidate the mechanisms underlying the spontaneous relaxation of contracted mesangial cells on collagen gel after exposure to LPA, we measured the accumulation of cAMP in monolayers exposed to LPA in the presence of the phosphodiesterase inhibitor IMX. As shown in Fig 9, basal cAMP levels were 9.0±0.3 pmol/mg protein. Three minutes after stimulation by 30 µmol/L LPA, cAMP accumulation had increased 3.2-fold (29.0±3.2 pmol/mg protein, P<.01). After 3 minutes, cAMP content gradually declined over time yet remained significantly elevated over the control value by 20 minutes. Conversely, without stimulation by LPA, cAMP levels at 3 minutes were 12.6±0.6 pmol/mg protein and did not change significantly over time (P>.05). These results indicate that LPA significantly stimulated the accumulation of cAMP in rat mesangial cells.

View larger version (15K): [in this window] [in a new window]   Figure 9. cAMP accumulation over time in response to LPA. Confluent monolayers of rat mesangial cells were stimulated with 30 µmol/L LPA () in the presence of 0.2 mmol/L IMX, and cAMP content was measured. Each point is the mean±SEM of triplicate dishes. *P<.05 vs unstimulated control cells (•).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The experiments reported in the present study constitute the first systematic characterization of the contractile properties of LPA in cultured rat mesangial cells. When mesangial cells are cultured on collagen gel and stimulated by LPA, they respond with a rapid and transient contraction. The contractile response, which we determined by using phase-contrast microscopy, delineated a significant decrease of CSA that occurred not only in the cell body but also included the cytoplasmic extensions (Fig 1). As demonstrated by electron microscopic examination of rat kidney mesangial cells, these cytoplasmic extensions of the glomerular mesangium contain an abundance of contractile actin filaments.²² The polymerization and condensation of these actin fibers is believed to play a vital role in mesangial cell function and consequently in the control of glomerular filtration.¹ In the present study, the contractile response of mesangial cells induced by LPA was completely abolished by pretreatment of the cells with cytochalasin B, an actin filament disassembling agent. Consequently, it is reasonable to conclude that the contraction of mesangial cells induced by LPA in our model involves the activity of the network of actin microfilaments.

Increases in [Ca²⁺]_i level have been established as constituting the principal component mediating the contraction of mesangial cells.²³ In the present experiment, we demonstrated a rise in [Ca²⁺]_i that was associated with mesangial cell contraction. As shown in Fig 3, LPA induced typical biphasic [Ca²⁺]_i waveforms in fura 2–loaded mesangial cells, displaying a sharp transient peak lasting for 30 to 60 seconds and a long sustained tonic phase. This waveform is a well-recognized pattern that is induced by the application of diverse and established agonists such as ET-1, Ang II, and arginine vasopressin.^{16 19} We further demonstrated that when mesangial cells were stimulated with LPA in the absence of Ca²⁺, the spike increase in [Ca²⁺]_i was only partially (25%) reduced, whereas the subsequent sustained increment of [Ca²⁺]_i was completely abolished. These results support the formulation that the spike increase in [Ca²⁺]_i results primarily from the intracellular release of Ca²⁺ stores and in part from extracellular influx of Ca²⁺ and that the sustained increase in [Ca²⁺]_i results predominantly from the influx of extracellular Ca²⁺ alone.¹⁹

In the present study, preincubation of rat mesangial cells with isradipine significantly reduced not only the basal levels of [Ca²⁺]_i but also the spike increase in [Ca²⁺]_i in response to LPA. The reduction of basal [Ca²⁺]_i levels by isradipine, together with previous reports that L-type (DHP-sensitive) Ca²⁺ channels are dependent on membrane voltage,^{24 25} strongly suggests a role for the expression of L-type Ca²⁺ channels in maintaining the resting state of mesangial cells. L-type Ca²⁺ channels have also been reported to maintain resting [Ca²⁺]_i levels in cultured rat aortic smooth muscle cells²⁶ and in intact isolated porcine coronary arteries.²⁷

Recent studies have also demonstrated the involvement of L-type Ca²⁺ channels in promoting contraction in several contractile cell types. Using the whole-cell patch-clamp technique, Baron et al²⁸ demonstrated that thrombin-induced contraction of portal vein smooth muscle cells was completely inhibited by isradipine. Using micropuncture techniques, Ichikawa et al²⁹ also reported that verapamil inhibited Ang II–induced changes in the glomerular ultrafiltration coefficient in the rat. They postulated that the effect on the glomerular ultrafiltration coefficient was attributable to the inhibition of mesangial cell contractility related to Ang II–induced Ca²⁺ entry. In contrast, studies in cultured mesangial cells by Takeda et al³⁰ reported that verapamil (50 µmol/L) failed to inhibit in vitro Ang II–induced changes in mesangial shape, although verapamil partially inhibited Ang II–induced Ca²⁺ influx. Similarly, Simonson and Dunn¹⁶ and Huang et al³¹ observed that although ET-1–stimulated Ca²⁺ entry was partially inhibited by either nifedipine (10 µmol/L) or manidipine (1 µmol/L), there was no inhibition of the contraction of cultured mesangial cells. Observations from our laboratory that were obtained when a lower dose of the Ca²⁺ channel blocker isradipine (0.5 µmol/L) was used also demonstrated a decrease in LPA-induced Ca²⁺ entry without inhibition of mesangial cell contraction. Nevertheless, a slight increase of the concentration of isradipine to 1 µmol/L was sufficient to block completely the LPA-induced contraction of rat mesangial cells cultured on collagen gel. Thus, the reasons for the discrepancies between these studies may relate to the differing experimental systems or to the potency of the Ca²⁺ antagonists that were used. Isradipine is a highly selective DHP-sensitive Ca²⁺ channel blocker³² and was 10-fold more potent than other DHP-sensitive Ca²⁺ channel antagonists in blocking growth factor-induced DNA synthesis in cultured rat smooth muscle cells.³³ These factors may account for the different results observed in the above-described studies of contractility. Our findings clearly indicate that DHP-sensitive Ca²⁺ channels play a crucial role in mediating the contractile response of mesangial cells by LPA.

Under the conditions of our contraction studies, stimulation with LPA (30 µmol/L) induced tachyphylaxis by desensitizing the mesangial cells to a subsequent identical challenge with LPA. The phenomenon was also confirmed by the Ca²⁺ mobilization studies, in which prior exposure of the cells to LPA reduced the subsequent responsiveness of the cells to further stimulation by LPA (Fig 5). This type of agonist-induced desensitization is a property shared by a number of receptors, including ß-adrenergic,³⁴ vasopressin, and arachidonic acid receptors.³⁵ In various signaling systems, agonist-induced attenuation of cellular responsiveness is an important autoregulatory phenomenon, and the occupancy of the receptor by the agonist appears to be critical. To date, protein kinase C, cAMP-dependent kinase, and receptor-specific receptor kinase have all been implicated in mediating agonist-induced desensitization.³⁶ In our experimental system, downregulation of protein kinase C by treatment with phorbol ester (12-O-tetradecanoylphorbol 13-acetate, 100 ng/mL) for 24 hours had no effect on homologous desensitization as assessed by Ca²⁺ mobilization in response to sequential stimulation of mesangial cells by LPA (data not shown). The observation of Jalink et al³⁷ that protein kinase C was not involved in the LPA-induced homologous desensitization in fibroblasts is consistent with our findings in mesangial cells. In conclusion, our findings in concert with the above-cited studies indicate that it is unlikely that protein kinase C is involved in the process of LPA-induced homologous desensitization in mesangial cells. Further studies examining homologous desensitization in mesangial cells will be required to elucidate the determinants of this phenomenon.

The gradual and spontaneous attenuation of the contractile response and subsequent relaxation of mesangial cells subjected to LPA prompted us to investigate whether the production of PGE₂ and the reactive accumulation of cAMP could account for this phenomenon. PGE₂ is a major vasodilatory metabolite of arachidonic acid in rat mesangial cells that have been stimulated by a variety of contractile stimuli including Ang II, arginine vasopressin, and ET-1.^{21 38} On the other hand, vasoconstrictor agonists such as leukotrienes C₄ and D₄ do not stimulate production of PGE₂.³⁹ Incubation of mesangial cells with exogenous PGE₂ causes a rapid rise in cAMP.⁴⁰ Consequently, stimulation of PGE₂ and the subsequent accumulation of cAMP is thought to constitute an important negative-feedback reaction to contraction. This phenomenon has been commonly observed not only in smooth muscle cells but also in glomerular mesangial cells.^{21 41} In concert with the observation that increased cAMP levels decrease the sensitivity of myosin light chain kinase to Ca²⁺ activation in smooth muscle cells,⁴² a PGE₂-mediated increase in cAMP might serve to regulate mesangial cell contraction.⁴⁰ In the present study, inhibition of cyclooxygenase with indomethacin blocked the spontaneous relaxation of LPA-contracted mesangial cells. In concert with the demonstration of an increased production of PGE₂ and cAMP accumulation within 5 minutes after the addition of LPA, these data suggest that an increase in these vasodilatory compounds may mediate the spontaneous relaxation of contracted mesangial cells in our experiments.

Although the precise mechanisms whereby PGE₂ production is stimulated by LPA have not been established, increasing evidence indicates that hormonally induced arachidonic acid release and subsequent prostaglandin production are mediated through the activation of PLA₂.⁴³ Recently, several mechanisms whereby PLA₂ is activated have been proposed: First, PLA₂ can be activated directly via a receptor-coupled G protein.^{8 44} Second, PLA₂ may be activated as a consequence of increased cytoplasmic calcium following phosphoinositide hydrolysis by PLC.⁴⁵ Third, PLA₂ may be stimulated by mitogen-activated protein kinase as proposed by Lin et al.⁴⁶ Hitherto, studies in cultured mesangial cells have suggested that PGE₂ production is linked to the stimulation of PLC and increased cytoplasmic Ca²⁺ in vasopressin-activated,⁴⁷ Ang II–activated,⁴³ or ET-1–activated cells.⁴⁸ In the present study, the activation of PLA₂ by LPA was manifested by an increase of arachidonic acid release. In addition, LPA-induced phosphoinositide hydrolysis was indicated by the production of IP₃ and [Ca²⁺]_i mobilization in the cultured mesangial cells. Collectively, it is tempting to suggest that LPA, in common with other Ca²⁺-mobilizing hormones, stimulates the PLA₂ signal transduction pathway, presumably through the hydrolysis of phosphoinositide by PLC, thereby enhancing the production of PGE₂.

Several lines of evidence provide a theoretical framework for postulating that our present findings have pathophysiological relevance. LPA has been identified in the sera in an albumin-bound form in concentrations of 2 to 20 µmol/L.^{11 49} Recent work by Tokumura and colleagues^{50 51} and Jalink et al,³⁷ who used concentrations of LPA in the 10- to 100-µmol/L range, have demonstrated that LPA induces contraction of intestinal and uterine smooth muscles from rats and exerts mitogenic properties in fibroblast and vascular smooth muscle cells. Our present demonstration that similar concentrations of LPA also induce contraction of rat mesangial cells may have important clinical implications regarding the pathogenesis of diverse renal disorders. It is well established that mesangial cells are separated from the glomerular capillary lumen by the fenestrated endothelium.^{2 52} Because of this anatomic configuration, the entry of LPA, presumably produced from aggregated platelets at the site of injury, is facilitated and can thereby modulate mesangial cell function and possibly contribute to the pathogenetic cascade.

Finally, recent evidence suggests that LPA may also act at the level of the mesangium in an autocrine manner. In a preliminary report, Wada et al¹³ have demonstrated that LPA was produced and released in mesangial cells when stimulated by group II PLA₂ to promote mesangial cell mitogenesis. Because PLA₂ is considered to play a role in the initiation and propagation of inflammatory reactions in mesangial cells,¹⁴ it is reasonable to speculate that PLA₂ may exert its effects on the glomerular microcirculation in part through LPA. In concert, our data suggest that LPA may contribute in both a paracrine and autocrine manner in the process of amplifying and propagating glomerular injury, thereby contributing to the pathogenesis of glomerulonephritis and renal dysfunction.

	Selected Abbreviations and Acronyms

 

Ang II = angiotensin II BSA = bovine serum albumin CSA = cell surface area DHP = dihydropyridine ET-1 = endothelin 1 IMX = 3-isobutyl-1-methylxanthine IP3 = inositol 1,4,5-tris-phosphate KHH = Krebs-Henseleit HEPES LPA = lysophosphatidic acid PAF = platelet-activating factor PDGF = platelet-derived growth factor PGE2 = prostaglandin E2 PLA2, PLC, and PLD = phospholipases A2, C, and D, respectively

	Acknowledgments

 
This study was supported in part by a research grant from the Kidney Foundation of South Florida. The authors would like to thank Parvaneh Safari for her technical assistance and Elsa V. Reina for her secretarial assistance.

Received March 16, 1995; accepted July 12, 1995.

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