Background

In inflammatory glomerular diseases, proliferation, as well as apoptosis of mesangial cells (MCs), has been shown histomorphologically. Both processes may regulate the cellular content of the mesangium by closely influencing each other. In the present study, we examined whether the cytoplasmic free Ca²⁺ concentration [Ca²⁺]_i is involved as a key second messenger in the regulation of proliferative and apoptotic events.

Methods

Thapsigargin, an inhibitor of the endoplasmic Ca²⁺-Mg²⁺-ATPase, was used as a test substance to investigate the role of [Ca²⁺]_i in signaling MC apoptosis and growth in vitro. Apoptosis was determined by nuclear chromatin staining with Hoechst 33258, by a [³H]-thymidine–based DNA fragmentation assay or by flow cytometry detecting binding of FITC-conjugated annexin V. Proliferation was measured by [³H]-thymidine incorporation into acid-precipitable material and corroborated by cell counting.

Results

Thapsigargin significantly induced apoptosis and inhibited proliferation dose dependently in nanomolar concentrations without evoking necrotic damage when administered not longer than 12 hours. Significant apoptosis was measurable after a six-hour treatment of MCs with thapsigargin. Determination of [Ca²⁺]_i by fura-2–dependent spectrofluorometry showed that thapsigargin was able to induce prolonged [Ca²⁺]_i rises that could be prevented by preincubation with the intracellular Ca²⁺ chelator 1,2-bis(2-aminophenoxy)-ethane-N,N,N', N'-tetra-acetic acid (BAPTA) acetomethyl ester (AM). BAPTA had no influence on MC viability but reversed thapsigargin-induced apoptosis to control levels. After thapsigargin treatment (100 nmol/L, 12 hours), apoptotic MCs had a significantly higher [Ca²⁺]_i of 251 25 nmol/L (N = 41) as compared with MCs that were not or not yet apoptotic ([Ca²⁺]_i of 116 20 nmol/L, N = 26, P < 0,05). Platelet-derived growth factor (PDGF), a well-characterized growth factor for MCs, reversed the effects of thapsigargin on proliferation and apoptosis in a similar fashion as BAPTA. PDGF acutely stimulated increases of [Ca²⁺]_i but abolished thapsigargin-dependent, but not angiotensin II- or ATP-induced Ca²⁺ rises when administered during a 12-hour preincubation.

Conclusions

Our data suggest that a sustained increase of [Ca²⁺]_i may serve as a signal to trigger MC apoptosis. Growth factors such as PDGF can abolish apoptosis induced by elevations of [Ca²⁺]_i by altering intracellular Ca²⁺ signaling.

METHODS

Reagents

Thapsigargin, angiotensin II, adenosine 5'-triphosphate (ATP), and 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetra-acetic acid-acetomethyl ester (BAPTA-AM) were obtained from Calbiochem (Bad Soden, Germany), PDGF-BB from Boehringer Mannheim (Mannheim, Germany), [³H]-thymidine (specific activity 5.0 Ci/mmol) from Amersham-Buchler (Braunschweig, Germany), and FITC-labeled annexin V from Boehringer Ingelheim Bioproducts (Heidelberg, Germany). Fura-2 AM and all other reagents were purchased from Sigma (Deisenhofen, Germany).

Animals and culture of glomerular mesangial cells

Mesangial cells, derived from 6- to 10-week-old male SD rats, were isolated, cultured, and characterized as described previously²¹. MC culture medium (CM) consisted of RPMI 1640 supplemented with 1 mmol/L L-glutamine, 50 U/mL penicillin, and 50 g/mL streptomycin (Seromed, Berlin, Germany). For the first two passages, CM also contained 5 g/mL bovine insulin, 5 mg/mL transferrin, and 5 ng/mL selenite. CM was supplemented with 0.5 to 10% fetal calf serum (FCS). For experiments, MCs between the third and fifth passages were exclusively used.

Quantitation of apoptosis by DNA fragmentation assay

Quantitation of DNA fragmentation was performed after labeling MC with [³H]-thymidine according to a method described by Higuchi and Aggarwal²².

Mesangial cells subcultured in 24-well dishes at a density of 3 10⁴ cells/mL in CM supplemented with 10% FCS were synchronized and slowed down in growth by reducing serum in CM to 5% FCS at day 2, to 2.5% FCS at day 3, and to 0.5% at day 4. Cells were then incubated with 3 Ci/mL [³H]-thymidine for 20 hours, washed extensively, and treated with the test substances for 1 to 20 hours. Afterward, MCs were again washed and postincubated in CM with 0.5% FCS for 12 hours in order to enable processing of apoptosis. CM was removed carefully and MCs solubilized in 1 mL hypotonic lysis buffer [0.5% Triton X-100, 5 mmol/L Tris, 20 mmol/L ethylenediaminetetraacetic acid (EDTA), pH 8] for 30 minutes on ice. Cell lysates were centrifuged at 13,000 g for 20 minutes to separate intact chromatin from fragmented DNA. Supernatants containing cleaved DNA and the pellets that were dissolved in 0.5 N NaOH were separately transferred for scintillation counting. For the total counts, MCs were lyzed by the addition of 20 L of 20% sodium dodecyl sulfate (SDS) without performing high-speed centrifugation. The percentage of cleaved DNA was calculated according to the following formula:

DNA fragmentation

= cpm of test sample X 100 total cpm

Baseline DNA fragmentation was scattered between 2 and 15% when MCs were cultured in medium alone. The percentage of increases or decreases of apoptosis by the tested substances in relationship to basal apoptosis (% of control) are depicted in the figures.

Cell necrosis was determined by trypan blue exclusion [10% trypan blue in phosphate-buffered saline (PBS)] or by low propidium iodide (PI) staining using flow cytometry (discussed later in this article).

Detection of apoptosis by annexin V binding

Mesangial cell binding of FITC-labeled annexin V indicating early stages of apoptosis was measured by flow cytometry. MCs cultured and treated as outlined previously in this article were trypsinized from culture flasks, washed extensively in ice-cold PBS, and incubated in PBS containing 2% bovine serum albumin (BSA) and 0.01% NaN₃ on ice to block unspecific binding of fluorochromes. Unfixed cells were then labeled with annexin V-FITC (2.5 g/mL in PBS containing 1.8 mmol/L CaCl₂) and PI (5 g/mL) for 15 minutes on ice before washing again in saline. MCs that exhibited low staining of nuclear chromatin by PI and high binding of annexin V were identified as apoptotic cells; high annexin V binding and PI staining indicated necrotic damage. Cells were analyzed on a FACScan flow cytometer (Becton Dickinson, Mountain View, CA, USA) for a total of 10,000 events.

Visualization of chromatin fragmentation

Mesangial cells (2 10³/mL) were seeded out to four-well glass chamber slides (Nunc, Wiesbaden, Germany) and synchronized as described previously in this article. After treatment with the test substances, cells were fixed in 4% paraformaldehyde in PBS for 10 minutes on ice, rinsed twice in PBS, and permeabilized with ice cold 80% ethanol for 5 minutes. The nuclear dye Hoechst 33258 was added at a concentration of 5 mol/mL for 20 minutes at 4°C. Thereafter, slides were rinsed three times in PBS and embedded in 5% glycerol for analysis by immunofluorescence microscopy. The percentage of apoptotic cells was determined by counting nuclei with condensed or fragmented chromatin after Hoechst 33258 staining from 100 MCs for each experiment.

Proliferation assay

Proliferation of subconfluent MCs was quantitated by determination of the incorporation of [³H]-thymidine. MCs (4 10⁴/mL) that were growth limited as described previously were washed twice with PBS and treated with test substances in CM with 0.5% FCS for 12 hours. For the last six hours of the incubation period, MCs were incubated with 3 Ci/mL [³H]-thymidine, and were washed and solubilized in 1 mL 0.1% SDS. Precipitation on ice was performed with 200 L of 20% trichloroacetic acid (TCA) overnight. Acid-precipitable material was pelleted by centrifugation, dissolved in 0.5 N NaOH, and transferred for liquid scintillation counting. Furthermore, MC growth was analyzed by counting cells microscopically.

Microspectrofluorometric measurement of [Ca²⁺]_i

Mesangial cells (5 10³/mL) were cultured on 35 mm glass cover slips and were slowed down in growth as described previously in this article. MCs were incubated with 1 mol/L of the [Ca²⁺]_i-sensitive dye fura-2 AM dissolved with surfactant Pluronic F-127 (0.1 g/L) for 30 minutes in Ham's F-12 medium (GIBCO BRL, Eggenstein, Germany) at 37°C as described previously²³. The measurements were performed in a constantly perfused (10 mL/min) chamber, where the cover slips were fixed. MCs were perfused with buffer containing 110 mmol/L NaCl, 25 mmol/L NaHCO₃, 10 mmol/L HEPES, 3.6 mmol/L KCl, 5 mmol/L glucose, 1 mmol/L MgCl₂, and 1.3 mmol/L Ca-gluconate for 20 minutes at 37°C before the experiments were started. MCs were exited at wavelengths of 340, 360, and 380 nm with a xenon-quartz lamp (XBO 75 W; Zeiss, Jena, Germany) using a filter wheel rotating at 10 Hz. Fluorescence was measured by a photon-counting tube (Hamamatsu H 3460-04, Herrsching, Germany) at 500 to 530 nm. Ten data points were averaged, resulting in a time resolution of 1 Hz. Fluorescence signals were taken from five MCs using an adjustable diaphragm. Autofluorescence was measured before loading cells with fura-2 and subtracted from the original data for each experiment. The experiments were analyzed and controlled with a PC-486 computer system and specific software (U. Fröbe, Physiologisches Institut, Universität Freiburg, Freiburg, Germany). At the end of each experiment, calibration of [Ca²⁺]_i was performed by incubation of cells with ionomycin (1 mol/L) in the presence (1.3 mmol/L) and absence of extracellular Ca²⁺ [buffered with 5 mmol/L egtazic acid (EGTA)]. Maximum and minimum values of the 340/380 nm excitation fluorescence ratio were used for all calibrations to estimate the [Ca²⁺]_i.

To determine whether thapsigargin-induced MC apoptosis was a direct consequence of [Ca²⁺]_i elevation, MCs stained by annexin V-FITC (2.5 g/mL for 15 minutes) and loaded with fura-2 AM were aimed visually by spectrofluorescence microscopy for [Ca²⁺]_i measurements. Annexin V-FITC–positive apoptotic cells and annexin V-FITC–negative intact cells were analyzed separately for [Ca²⁺]_i.

Statistics

Data are presented as mean values SEM. The number of experiments (N) is depicted within the figure bars. One-way analysis of variance (ANOVA) test (Microcal Origin 5.0 Software, Northhampton, MA, USA) was used for statistical analysis. P values of less than 0.05 were considered significant. For statistics and depiction of data in the figures, changes of the level of apoptosis or proliferation of MC by the agonists are expressed in percentage taking control medium as 100% because baseline apoptosis scattered between 2 and 15%.

RESULTS

Effects of thapsigargin on MC apoptosis and proliferation

As shown in Figure 1a, the Ca²⁺-ATPase inhibitor thapsigargin induced apoptosis in nanomolar concentrations dose dependently when MCs were treated for 12 hours. A thapsigargin concentration of 100 nmol/L increased apoptosis by 138 30% above medium control. Time kinetic studies revealed that a significant increase of MC apoptosis was demonstrable at minimum incubation times of six hours Table 1. At incubation times above 12 hours, necrosis rates above 10% were found (12 3% necrotic MC after 20 hours of treatment with thapsigargin; Table 1).

Figure 1.

Dose-dependent effects of thapsigargin on mesangial cell (MC) apoptosis (A) and proliferation (B). MCs were synchronized and limited in growth by reducing serum concentration of the culture medium (CM), as described in the Methods section, before being treated with thapsigargin for 12 hours. Apoptosis was determined by a [³H]-thymidine–based DNA fragmentation assay. Means SEM are given taking control as 100%. At least six independent experiments were done, with the total number of cultures indicated within the graph bars. *P < 0.05 compared with control.

Full figure and legend (28K)

Table 1 - Determination of cell counts, apoptosis and necrosis of mesangial cells.

Full table

In the following experiments, thapsigargin was administered at a concentration of 100 nmol/L for 12 hours if not otherwise indicated. Furthermore, thapsigargin significantly reduced MC proliferation at concentrations and incubation times that were chosen for apoptosis studies Figure 1b. Experiments with flow cytometry detecting phosphatidylserine exposure of apoptotic cells by annexin V staining showed that under control conditions, 3 2% of MC bound annexin V, whereas after treatment with thapsigargin (100 nmol/L for 12 hours), 20 4% of cells exerted an increased annexin V staining, indicative for apoptosis (N = 12, P < 0.05). Representative flow cytometry tracings after labeling MCs with annexin V-FITC for apoptosis detection and PI for necrosis detection are given in Figure 5 (upper panel). Staining MCs with the nuclear dye Hoechst 33258 as a further method to confirm apoptosis in later stages revealed significant chromatin condensation and fragmentation only in MCs treated with thapsigargin for at least six hours Table 1. Figure 2 provides typical images of intact or late apoptotic MCs treated for 12 hours with 100 nmol/L thapsigargin after Hoechst staining.

Figure 5.

Induction of MC apoptosis by thapsigargin (100 nmol/L, 12 hours) and reversal of apoptosis by pretreatment of cells with BAPTA-AM (BAPTA, 10 mol/L, 30 minutes) indicated by flow cytometry. In this multiparameter flow cytometry, low nuclear staining by PI excluded significant necrotic cell damage (8%). Apoptosis was determined by binding of FITC-labeled annexin-V. Percentages of the cells within the four quadrants are depicted with the number in the lower right quadrant indicating apoptotic cells. A representative experiment out of three is shown.

Full figure and legend (67K)

Figure 2.

Representative immunofluorescence micrographs of mesangial cells (MCs) treated without (A) or with thapsigargin (B, 100 nmol/L for 12 h). Nuclear staining was performed with the Hoechst 33258 dye, which gave a homogeneous chromatin architecture for control cells but intensive nuclear condensation and fragmentation after thapsigargin treatment. Representative images out of six experiments are shown.

Full figure and legend (160K)

Effects of thapsigargin on [Ca²⁺]_i and abrogation of apoptosis by Ca²⁺ buffering

Figure 3 shows that thapsigargin (100 nmol/L) markedly elevated [Ca²⁺]_i of MCs as presented by the 340/380 fluorescence ratio. This increase of [Ca²⁺]_i was completely abrogated when MCs were preincubated with the intracellular Ca²⁺-chelator BAPTA-AM (10 mol/L) for 30 minutes. These data are summarized in Figure 7c. Removing Ca²⁺ from extracellular medium by addition of EGTA (1 to 5 mmol/L) and culturing MCs in nominally Ca²⁺-free medium highly diminished the adherence of MCs so that MC apoptosis was not measurable under these conditions. However, detecting [Ca²⁺]_i of MCs after removal of Ca²⁺ in the presence of EGTA (5 mol/L) gave thapsigargin-induced [Ca²⁺]_i elevations that were similar as for cells left in the presence of Ca²⁺ ([Ca²⁺]_i 121 34 nmol/L in Ca²⁺-free solution, N = 6, vs. 78 36 nmol/L, N = 19; lower panel of Figure 3).

Figure 3.

Effect of thapsigargin on [Ca²⁺]_i of untreated MC (-BAPTA) or MC treated with BAPTA-AM (+BAPTA, 10 mol/L) for 30 minutes and effect of external Ca²⁺ removal on thapsigargin-induced [Ca²⁺]_i. Representative tracings of fura-2 fluorescence ratios out of six independent experiments are given. External Ca²⁺ depletion was achieved by superfusing cells in Ca²⁺-free solution supplemented with 5 mmol/L EGTA.

Full figure and legend (19K)

Figure 7.

Effect of PDGF on MC [Ca²⁺]_i after acute administration (1.25 nmol/L, upper panel of A) and abrogation of the effect of thapsigargin on [Ca²⁺]_i after pretreating MCs with PDGF for 12 hours (lower panel of A). (B) The increases of [Ca²⁺]_i by angiotensin II (1 mol/L) and ATP (100 mol/L) but the lacking effect of thapsigargin (100 nmol/L) when MCs were preincubated with PDGF (1.25 nmol/L) for 12 hours. (C) The effects of PDGF on [Ca²⁺]_i after an acute administration of PDGF and of thapsigargin (TG) without or with pretreatment of MC by BAPTA-AM (10 mol/L, 30 min; TG + BAPTA) or PDGF (1.25 nmol/L, 12 hours; TG + PDGF) are summarized. (A and B) Representative tracings depicting 340/380 ratios of fura-2 fluorescence levels out of four independent experiments are shown. (C) Means SEM are given presenting the numbers of cultures within the graph bars. *P < 0.05 as compared with control; #P < 0.05 as compared with TG alone.

Full figure and legend (35K)

Application of BAPTA-AM for 30 minutes did not alter the baseline DNA fragmentation of MCs, but reversed the thapsigargin-induced increase of apoptosis back to control levels. This was shown by the [³H]-thymidine–based DNA fragmentation assay Figure 4, flow cytometry detecting annexin V binding Figure 5, as well as by Hoechst 33258 staining Table 1. These data suggest that thapsigargin evoked apoptosis of MCs by leading to an increase of [Ca²⁺]_i consecutive to a release from internal cytoplasmic stores and inducing secondary Ca²⁺-dependent mechanisms responsible for DNA degradation.

Figure 4.

Induction of apoptosis by thapsigargin (TG; 100 nmol/L, 12 h) of MCs pretreated without or with BAPTA-AM (10 mol/L, 30 min). BAPTA-AM alone did not alter basal apoptosis significantly, but reversed thapsigargin-induced apoptosis to control levels. Means SEM are given with the numbers of cultures indicated within the graph bars. *P < 0.05 as compared with control; #P < 0.05 as compared with TG.

Full figure and legend (13K)

[Ca²⁺]_i of MCs treated with thapsigargin in intact and apoptotic cells

Since not all MCs treated with thapsigargin showed features of apoptosis, we examined whether differences of basal [Ca²⁺]_i were measurable between intact (annexin V negative) and apoptotic (annexin V positive) cells. Intact thapsigargin-treated MCs had a significantly lower [Ca²⁺]_i of 116 20 nmol/L (N = 26) as compared with apoptotic thapsigargin-treated cells, which had a [Ca²⁺]_i of 251 25 nmol/L (N = 41, P < 0.05).

Effects of PDGF on proliferation and apoptosis and counter-regulation of the action of thapsigargin

The well-characterized growth factor PDGF dose dependently induced proliferation of MCs as determined by thymidine incorporation at nanomolar concentrations when cells were treated for 12 hours Figure 6a. Cell counting corroborated MC growth by PDGF Table 1. Preincubation with PDGF (1.25 nmol/L) for 12 hours and consecutively exposing cells to thapsigargin (100 nmol/L) for further 12 hours normalized MC growth to basal levels and reversed the reduction of proliferation induced by thapsigargin Figure 6b. At lower PDGF concentrations of 0.25 nmol/L, this treatment had no influence on basal MC apoptosis, but reversed the thapsigargin-induced increase of apoptosis back to baseline. At higher PDGF concentrations (1.25 nmol/L), PDGF per se reduced the basal MC DNA fragmentation rate Figure 6c.

Figure 6.

A dose-dependent induction of MC proliferation by PDGF (A), effects of PDGF (1.25 nmol/L, 12-h preincubation) on MC proliferation in response to thapsigargin (TG; 100 nmol/L 12 h; B) and on TG-induced apoptosis using the indicated low or high PDGF concentrations (C). PDGF was able to reverse the reduced MC proliferation in response to TG to control levels (B) and to antagonize TG-induced apoptosis (C). Means SEM are given. The numbers of cultures are indicated within the graph bars. *P < 0.05 as compared with control; #P < 0.05 as compared with TG alone.

Full figure and legend (39K)

Effects of PDGF on [Ca²⁺]_i and thapsigargin-induced [Ca²⁺]_i increases

Treatment of MCs with PDGF led to an acute increase of [Ca²⁺]_i, as shown by the 340/380 fluorescence ratios. However, preincubation with PDGF for 12 hours and then exposing cells to thapsigargin abolished the rise of [Ca²⁺]_i completely. Figure 7a gives original tracings of representative experiments. Figure 7c summarizes the acute effects of thapsigargin (100 nmol/L) and PDGF (1.25 nmol/L) on [Ca²⁺]_i of MCs and compares them with the lack of effects of thapsigargin when MCs were preincubated with BAPTA-AM for 30 minutes or PDGF for 12 hours. Interestingly, angiotensin II and ATP, both well-known agonists leading to [Ca²⁺]_i rises of MCs, were, in contrast to thapsigargin, still able to evoke Ca²⁺ peaks after a 12-hour preincubation with PDGF. A representative tracing is shown in Figure 7b. Angiotensin II (1 mol/L) elevated [Ca²⁺]_i from 143 18 to 396 38 nmol/L (N = 5) after preincubation of MCs with PDGF (1.25 nmol/L), which was similar to MCs without PDGF pretreatment (data not shown). Simultaneous administration of PDGF and thapsigargin to MCs induced a significant rise of [Ca²⁺]_i without exerting additive or potentiating effects (data not shown).

DISCUSSION

The endoplasmic reticular Ca²⁺-ATPase inhibitor thapsigargin was used in the present study to investigate whether an increase of [Ca²⁺]_i can serve as a signal to initiate MC apoptosis. Many previous studies have shown that a rise of [Ca²⁺]_i by thapsigargin involves an initial release of the cation from microsomal, nonmitochondrial Ca²⁺ pools, which induces and is followed by a capacitative Ca²⁺ influx from the extracellular space^19,20. Our results confirmed that thapsigargin elevated [Ca²⁺]_i of MCs also in nominally Ca²⁺-free medium. In contrast to Ca²⁺ ionophores, which permeabilize cell membranes and organelle membranes unspecifically, thapsigargin is a suitable and specific test substance to examine the significance of Ca²⁺-signaling in apoptosis^24,25. With respect to increasing [Ca²⁺]_i, it mimics the action of various natural mediators such as vasopressor agonists (for example, endothelin, angiotensin II), growth factors (for example, PDGF, basic fibroblast growth factor) or cytokines (for example, interleukin-2)^19,23,26.

Our data show that treatment with thapsigargin resulted in apoptosis of MCs, which were in a synchronized and growth-limited state. MCs kept in a proliferative state by serum supplementation (>1% FCS in medium) were resistant to the apoptosis-inducing effects of thapsigargin. We detected features of apoptosis by three different techniques; this study shows that thapsigargin induced nuclear chromatin condensation and fragmentation by Hoechst 33258 nuclear staining as well as plasma membrane alterations indicative for apoptosis using annexin V-based flow cytometry. An increase of [Ca²⁺]_i was an essential trigger to activate apoptotic processes of MCs because intracytoplasmic chelation of Ca²⁺ by BAPTA-AM completely antagonized the effects of thapsigargin and reversed apoptosis to basal levels. Furthermore, MCs treated with thapsigargin showed significantly higher [Ca²⁺]_i when they were identified as apoptotic cells by annexin V binding as compared with MCs that were still intact and negative for annexin V binding. When MCs were treated with thapsigargin for more than 12 hours, progression of cells into necrotic stages took place, but the percentage of apoptotic MCs never exceeded one third Table 1. MCs that peaked with [Ca²⁺]_i were identified as senescent cells, but it is not clear by which mechanisms the surviving cells quenched [Ca²⁺]_i elevations and were rescued from apoptosis. Probably, MCs that locally produce high levels of autocrine growth factors such as PDGF are protected against apoptosis safely.

Thymocytes and lymphocytes are cells in which apoptosis in response to a rise of [Ca²⁺]_i could be first demonstrated^27,28. This was similarly achieved by Ca²⁺ ionophores, thapsigargin, and dexamethasone²⁹. Since all of these substances increased [Ca²⁺]_i, it was suggested that Ca²⁺ is a second messenger signal generally engaged as a trigger of apoptosis^17,20. In nonlymphoid blood cells such as neutrophils or monocytes, however, elevations of [Ca²⁺]_i retarded and antagonized apoptotic processes^18,30, so that [Ca²⁺]_i peaks are not able to initiate apoptotic pathways per se. It is striking that in lymphocytes or lymphoid cell lines, all exerting a high proliferative capacity, increases of [Ca²⁺]_i trigger apoptosis, whereas in neutrophils or monocytes, cells that are terminally differentiated and not or hardly not able to proliferate, a rescue from apoptosis takes place¹⁷. MCs that divide in culture and also significantly proliferate in vivo during different glomerular diseases fit into this scheme by initiating apoptosis after elevation of [Ca²⁺]_i.

This linkage between proliferation of cells and sensitivity to Ca²⁺ signal-dependent apoptosis prompted us to test the effects of the growth factor PDGF on thapsigargin-induced MC apoptosis. PDGF acutely caused a marked increase of [Ca²⁺]_i as it has been shown in several previous studies^31,32. In comparison to thapsigargin, [Ca²⁺]_i increases after PDGF exposure were similar with respect to height and duration, so we can widely exclude that the pattern of Ca²⁺ peaking determines whether a proliferative or an apoptotic pathway will be initiated. PDGF induced MC proliferation and using higher concentrations (1.25 nmol/L) even suppressed basal apoptosis. When MCs were pretreated with PDGF for 12 hours and were consequently exposed to thapsigargin, Ca²⁺-triggered apoptosis disappeared. Most interestingly, pretreatment with the growth factor exclusively abolished thapsigargin-induced elevations of [Ca²⁺]_i but not angiotensin II- or ATP-induced Ca²⁺ fluxes. These data indicate that increases of [Ca²⁺]_i per se are not sufficient to induce apoptosis or proliferation, although during both cellular processes, Ca²⁺ signaling occurs. Depletion of cytosolic Ca²⁺ stores by growth factors could be excluded since agonists were still able to induce [Ca²⁺]_i peaks after PDGF pretreatment.

Growth factors induce their effects via cell membrane receptors, which are coupled to protein tyrosin kinases. During the downstream events, phospholipase C- and phosphatidylinositol 3 kinase are involved, which generate rises of [Ca²⁺]_i, mobilization of transcription factors, and nuclear expression of protooncogenes^32,33,34. By the activation of these cascades, stimulation of Ca²⁺-dependent endonucleases, phospholipase A, or proteases engaged in apoptotic pathways³⁵ must be blocked by yet unknown mechanisms. Our results suggest that growth factors can alter Ca²⁺ signaling and cell survival depending on the duration of time they are present at sufficient concentrations.

In conclusion, our study shows that Ca²⁺ signals are involved in MC proliferation as well as in apoptosis and might serve as a regulatory link. Mediators such as PDGF support not only cell growth, but rescue MCs from apoptosis as survival factors by altering Ca²⁺ homeostasis.

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Acknowledgments

This study was supported by the IZKF Universität Münster, grant B5, Münster, Germany.