Apoptosis 2005; 10: 1457–1467


C 2005 Springer Science + Business Media, Inc. Manufactured in The Netherlands. DOI: 10.1007/s10495-005-1402-5

Alkaline stress-induced apoptosis in human pulmonary artery endothelial cells M. Cutaia, A. D. Black, I. Cohen, N. D. Cassai and G. S. Sidhu Pulmonary Disease Section, Department of Medicine, Veterans Administration Medical Center, Brooklyn Campus, SUNY/Downstate Health Sciences Center (M. Cutaia, A. D. Black, I. Cohen); Department of Pathology, Veterans Administration Medical Center, Manhattan Campus ( N. D. Cassai, G. S. Sidhu); Department of Pathology, NYU School of Medicine ( G. S. Sidhu)

Published online: 3 October 2005 The effect of alkaline stress, or an increase in extracellular pH (pHext), on cell viability is poorly defined. Human pulmonary artery endothelial cells (HPAEC) were subjected to alkaline stress using different methods of increasing pHext. Viability and mode of cell death following alkaline stress were determined by assessing nuclear morphology, ultrastructural features, and caspase-3 activity. Incubation of monolayers in media set to different pHext values (7.4–8.4) for 24-h induced morphological changes suggesting apoptosis (35–45% apoptotic cells) following severe alkaline stress. The magnitude of apoptosis was related to the severity of alkaline stress. These findings were confirmed with an assessment of ultrastructural changes and caspase-3 activation. While there was no difference in the intracellular calcium level ([Ca2+ ]i ) in monolayers set to pHext 7.4 versus 8.4 following the first hour of alkaline stress, blockade of calcium uptake with the chelator, EGTA, potentiated the magnitude of apoptosis under these conditions. Potentiation of apoptosis was reduced by calcium supplementation of the media. Finally, alkaline stress was associated with an increase in intracellular pH. This is the first report of apoptosis following alkaline stress in endothelial cells in the absence of other cell death stimuli.

Keywords: alkaline stress; apoptosis; cell death; human pulmonary artery endothelial cells.

Introduction Alterations in pH modify a diverse group of cellular functions, including cell proliferation and differentiation, enzyme activity, and cytokine and mediator release.1–6 Recent evidence suggests an important role for changes in pH in modifying the events in cell death pathways, but the findings are cell-type dependent. Prior work demonstrated that intracellular acidification is an early event in the apoptotic signaling cascade in some cell types,7 and Correspondence to: M. Cutaia, VA Medical Center, 800 Poly Place, Brooklyn, NY 11209-7104, USA. Tel.: +(718)-836-6600, Ext.: 6084, 6963; Fax: +(718)-630-2981; e-mail: [email protected]

that caspase activation triggers this acidification in several models.8–10 Nevertheless, the role of acidosis in apoptotic signaling remains controversial in view of contrasting findings among cell types. For example, extracellular acidosis or inhibition of the activity of pH-regulating systems, such as the Na+ /H+ exchanger (NHE), can prevent or inhibit the development of apoptotic or necrotic cell death in other models.11–15 In contrast, little is known about the effect of extracellular or intracellular alkalinization (“alkaline stress”) on viability or apoptotic signaling in most cell types. Alkaline stress is associated with cytotoxicity in several cell types.16,17 Recent work suggested that an increase in pHi is an “early” signaling event in several models of apoptosis.12,18,19 Our objective was to determine the effect of alkaline stress on viability and the mode of cell death in human pulmonary artery endothelial cells (HPAEC) in the absence of other cell death signals, and determine the role of altered calcium uptake in the observed changes in viability under these conditions. Recent evidence suggests that alkalosis worsens ventilatorinduced lung injury,17 but the specific cell types involved could not be identified in this work at the organ-system level. A more complete understanding of the effect of altered pH and alkaline stress on lung endothelium may have relevance to the pathogenesis and treatment of lung injury.

Materials and methods Cell culture Human pulmonary artery endothelial cells (HPAEC; Cascade Biologics, Portland, OR) were maintained in Medium 200 with low serum growth supplement (Cascade) and 10% FBS (Gibco, Carlsbad, CA). Cells were seeded onto 60-mm2 dishes for imaging experiments, 35-mm2 dishes for ultrastructure experiments, 48-well microplates for measuring caspase-3 activity, or Apoptosis · Vol 10 · No 6 · 2005

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24-well microplates for measuring intracellular pH with BCECF.

dia containing the various concentrations of EGTA noted above (298 versus 307 ml Os/L in media without EGTA versus media containing EGTA, 5 mM, respectively).

Experimental protocol for alkaline stress The pH of the media (pHext) was altered to induce alkaline stress in HPAEC monolayers using two different methods. In the first set of experiments, a CO2 independent media (Gibco) was adjusted to the desired pH (7.4–8.4) with HCl or NaOH before starting the experiment. Once adjusted, the media maintained a stable pHext for up to 72 h (data not shown). Alkaline stress was also induced by changing the ambient carbon dioxide (pCO2 ) concentration of the incubator from the normal 5%CO2 concentration to 0%, while maintaining cells in bicarbonate-buffered media, similar to a previously described method.4 Using both of these methods, alkaline stress was induced in near-confluent (∼75–90%) endothelial cell monolayers for varying time periods (1, 6, 24 h), followed by assessment of viability and mode of cell death, ultrastructural changes, caspase-3-like activity, or determination of intracellular pH, as described below.

Assessment of viability with fluorescent assay (Hoechst/PI) Viability and mode of cell death were assessed using an inverted microscope (Olympus IX70) equipped with a xenon light source (75 W) in monolayers loaded with Hoechst 33342 (10 µM) and propidium iodide (PI; 30 µM), as previously described.11 Cells were visualized under epifluorescence illumination using the appropriate filters and wavelength settings. Images of three to four fields per condition were analyzed using MetaMorph software (Universal Imaging, Chester, PA). Cells were scored as normal, apoptotic, or necrotic, as previously described.11 A minimum of 200 cells were counted per experimental condition. The data are presented as the percentage of apoptotic cells (% apoptotic cells), unless otherwise indicated. A similar set of viability experiments were performed following blockade of calcium uptake from the media in monolayers subjected to alkaline stress by incubating monolayers in media containing the calcium chelator, EGTA (1,2,5 mM-final concentration), followed by an assessment of changes in viability one hour later. Alkaline stress was induced by incubating monolayers in media pHext 8.4, while control monolayers were incubated in media pHext 7.4. Additional experiments were performed in which the media was supplemented with calcium (addition of CaCl2 , 2 mM) in the presence or absence of EGTA under the same conditions. Media osmolality demonstrated minimal change in osmolality in me1458 Apoptosis · Vol 10 · No 6 · 2005

Measurement of intracellular calcium concentration [Ca2+ ]i Measurement of the intracellular cytosolic calcium concentration [Ca2+ ]i was accomplished using fluo-3, as previously described.20 Monolayers seeded into 24-well plates were loaded with the probe at room temperature (27◦ C) by incubating the cells in HEPES-buffered MEM containing fluo-3 (2–5 µM), and probenecid (5 mM) to inhibit probe efflux. Fluo-3 was mixed one to one by volume with a 20% (w/v) solution of Pluronic-127 in DMSO before addition to the media to aid in solubility and dispersal of the probe. Monolayers were then incubated for an additional 20 min in the same media at 37◦ C to increase hydrolysis of the ester portion of the probe, followed by removal and addition of new media in each well before starting the experiment. Monolayers were visualized with an imaging system containing the following components: an inverted microscope (Olympus IX70; Objective: 20x) equipped with a mercury light source (75 W) linked to a CCD camera and computer-controlled camera shutter. Images were obtained with excitation/emission/dichroic filters (excitation: 505 nm; emission: 530 nm) placed in the light path. We monitored intracellular fluorescence, represented as the average gray scale value, continuously at 5 min intervals in control versus treated monolayers for one hour. The magnitude of the probe signal from all cells in each microscopic field was quantitated using the imaging software (MetaMorph, Universal Imaging; West Chester, PA).

Transmission electron microscopy (TEM) After exposure to alkaline stress for either 6 or 24 h, the monolayers were rinsed with PBS and fixed in place with 3% phosphate buffered glutaraldehyde (Electron Microscopy Sciences, Fort Washington, PA) pH 7.3 for 1 h at room temperature. Fixation of cells in place reduced artifacts, and maintained typical endothelial morphology better than fixing after detaching cells (data not shown). Cells were then loosened by scraping, collected in a microfuge tube, and pelleted. The pellets were fixed in the same fixative for several days. They were then washed with 0.1 M Sorensen’s phosphate buffer (Fisher Scientific, Fair Lawn, NJ), pH 7.3, twice at room temperature. Post-fixation was done with 2% osmium tetroxide (Electron Microscopy Sciences) for 1 h. The pellet was then dehydrated through a graded series of ethanols starting with 70% for 1 wash of 15 min, 95% for 3 washes of

Alkaline stress-induced apoptosis in human pulmonary artery endothelial cells

15 min, and 100% for 4 washes of 15 min each, then in propylene oxide (Electron Microscopy Sciences) for 2 washes of 10 min each. The pellet was infiltrated with a 1:1 mixture of propylene oxide and Embed-812 (Electron Microscopy Sciences), and rotated overnight on a Model 151 rotator (Scientific Industries, Bohemia, NY). The following day the pellets were transferred to a straight Embed-812 mixture and rotated for another 8 h. The samples were embedded in 00 size Beem capsules (Electron Microscopy Sciences) at 60◦ C. The resulting blocks were cut at 70 mm and sections were stained with uranyl acetate and lead citrate. They were examined in a JEM 1010 Electron Microscope (JEOL, Peabody, MA). At least ten grid hexagons were chosen at random and examined for each condition. Cells were categorized as viable, apoptotic, or necrotic based on standard criteria.21

Measurement of caspase-3-like activity The protocol from the manufacturer (EnzChek Caspase3 Assay Kit #2, Molecular Probes) was adapted for use on adherent cells in a 48-well microplate using a microplate fluorimeter (Gemini SpectraMaxXS, Molecular Devices), as previously described.11 Staurosporine (1 µM) was used as a positive apoptosis control. Following alkaline stress for 6 or 24-h, caspase was liberated from the cells by adding lysis buffer to each well following aspiration of the cell media, and incubating the plate on ice for 30 min. A reaction buffer containing the caspase3 substrate Z-DEVD-R110 was added. This substrate also binds caspase-7, making the assay a measure of caspase-3-like activity. After a 30-min incubation at room temperature, the plate was read in a fluorimeter set to 496ex/520em at intervals of 30 min for 6–8 h, with peak fluorescence obtained at six hours. Incubation of monolayers in media set to different pH values had no effect on the magnitude of the fluorescent signals during the assay because the media in each well was replaced with lysis buffer at pH7.4 at the end of the treatment period prior to performing the assay. The data are expressed as relative fluorescent units (RFU). Each experimental condition was replicated in three separate wells during an experiment.

Measurement of intracellular pH Intracellular pH was measured with the pH-sensitive fluoroprobe 2 ,7 -bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF; Molecular Probes) using an approach adapted from our prior work.22 Measurement of intracellular pH was done using a microplate fluorimeter capable of making dual excitation measurements (SpectraMax Gemini XS, Molecular Devices). Cells were loaded with BCECF (10 µM) in MEM at room temperature, followed by place-

ment in CO2 -independent media set to varying pH values (7.4–8.4) for 2 h at 37◦ C. The fluorimeter was set to capture the emission fluorescent signals at 535 nm, following alternating dual excitation at 490 and 440 nm, respectively, at the end of the 2 h incubation period. The data are expressed as the ratio of emission signals obtained following excitation at 490 nm (pH sensitive wavelength) and 440 nm (pH insensitive wavelength) in standard fashion.

Results Incubation of monolayers in CO2 -independent media set to different pH values for 24-h produced a significant loss of viability in HPAEC via apoptosis as indicated by the nuclear morphological changes observed with the Hoechst/PI assay (Figure 1). There were few apoptotic cells observed at media pHext values of 7.4 or 7.6. In contrast, the nuclear morphological changes observed following 24-h of alkaline stress were substantial at the higher media pH values (>7.6–8.0), demonstrating the typical alterations of nuclear morphology (chromatin condensation, nuclear ruffling, reduced nuclear size) observed in apoptosis. The percentage of apoptotic cells measured with the Hoechst/PI assay increased in direct proportion to the degree of alkaline stress, reaching a maximum of 36.1% at media pHext 8.4 (Figure 1). There was minimal necrosis following this exposure as indicated by the absence of typical morphological changes indicative of necrosis using this assay-ie. intact membrane integrity indicated by the lack of PI uptake in the cells. These findings were confirmed using a second method to induce alkaline stress by altering the ambient pCO2 concentration from 5% CO2 to 0%, while maintaining cells in bicarbonate-buffered media (Figure 2). Using this method, cells subjected to alkaline stress in an atmosphere of 0% CO2 for 6 h, which resulted in media pHext 8.44, demonstrated a small degree of apoptosis (∼8%) based on alterations of nuclear morphology. In contrast, alkaline stress for 24 h markedly increased the percent of apoptotic cells from 7.1 to 48.4%, in the presence of media pHext 8.49. Thus, these findings confirmed the results using CO2 -independent media (Figure 1), suggesting that the magnitude of apoptosis correlates with the duration and magnitude of alkaline stress. Examination of ultrastructure by transmission electron microscopy (TEM) following alkaline stress (6, 24– h) also revealed features typical of apoptosis (Figure 3, Tables 1 and 2). These ultrastructural changes were more numerous following a 24-h alkaline stress, and consisted of shrunken cells with dense cytoplasm, pyknotic chromatin packed against the nuclear membrane in smooth masses, and cytoplasmic budding. In addition, cells in the late stage of apoptosis were observed with breakdown of the nucleus into pyknotic nuclear fragments and the Apoptosis · Vol 10 · No 6 · 2005

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M. Cutaia et al. Figure 1. The effect of a 24-h alkaline stress in CO2 -independent media on viability and mode of cell death using the Hoechst/PI assay. A: Data represent the averaged % apoptotic nuclei ± S.E; n ≥ 200 cells per condition, taken from four fields; ∗ p < 0.01 versus control monolayers at pHext 7.4. B: Nuclear staining (Hoechst 33342) of cells maintained at pHext 7.4 for 24 h. C: Nuclear staining (Hoechst 33342) of cells maintained at pHext 8.4 for 24 h. Arrows indicate apoptotic nuclei.

formation of apoptotic bodies (Figure 3). The percent of apoptotic cells, based on these ultrastructural changes, increased with the magnitude of alkaline stress, from 2.5% in pHext 7.4 to 35% in pHext 8.4 (Table 1). Necrosis, distinguished by swelling and eventual rupture of the nucleus and organelles (Figure 3D) occurred at an equivalent low level in all samples set to different pHext values (≤4%; Table 1). Of note, a 6-h period of alkaline stress also induced apoptosis, based on a similar assessment of ultrastructural features, but at a significantly lower level at each pHext value (Table 2), compared to the findings at 24-h. Thus, the ultrastructural findings correlated with the assessment of morphological changes suggestive of apoptosis under the same experimental conditions and time points. Caspase-3-like activity, a classic marker of apoptosis, increased in response to extracellular alkaline stress of 6 or 24 h duration (Figure 4). Following 6 h of alkaline stress, a significant increase occurred in the wells treated with pHext 7.8, 8.0, and 8.4; the greatest increase was

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observed in media with pHext 8.0. A similar pattern was observed with the 24-h treatment. The increase in caspase-3 activity during alkaline stress was comparable to the increase observed following treatment with a wellknown inducer of apoptosis (staurosporine) in mammalian cells. Measurement of [Ca2+ ]i demonstrated no significant difference in the [Ca2+ ]i level at the start of the experiment or at the end of the 60-min measurement period between monolayers set to pHext 7.4 or pHext 8.4. This experiment was repeated several times (n = 5). The averaged results from these experiments, which include ∼125– 150 cells in each experimental condition, are presented in Figure 5. These findings indicate that alkaline stress does not induce a significant change in [Ca2+ ]i at this one hour time point, compared to untreated control monolayers. The one hour time point was chosen for evaluation based on the known difficulties of monitoring [Ca2+ ]i with a fluorescent probe (leakage of probe from cells) in cells undergoing a loss of viability, which occurs with

Alkaline stress-induced apoptosis in human pulmonary artery endothelial cells Figure 2. The effect of 6, 24-h alkaline stress induced by altered p CO2 on viability and mode of cell death using the Hoechst/PI assay. Data represent the averaged % apoptotic nuclei ± S.E.; n ≥ 200 cells per condition taken from four fields; ∗ p < 0.01 versus control at same time.

Table 2. The effect of a 24-h alkaline stress on viability and mode of cell death assessed with transmission electron microscopy (TEM). Cells were classified as viable, apoptotic, or necrotic based on standard criteria. pH ext

Table 1. The effect of a 6-h alkaline stress exposure on viability and mode of cell death assessed with transmission electron microscopy (TEM). Cells were classified as viable, apoptotic, or necrotic based on standard criteria. % Apoptotic cells

% Necrotic cells

pH ext

% Viable cells

7.4 (n = 125)

98.4

0

1.6

7.6 (n = 74)

97.2

1.4

1.4

7.8 (n = 91)

91.3

7.7

1.0

8.0 (n = 59)

86.4

11.0

1.7

8.4 (n = 129)

85.0

11.6

3.1

increasing frequency at later time points in monolayers subjected to alkaline stress. In contrast, blockade of calcium uptake in cells subjected to alkaline stress for one hour demonstrated a significant change in viability (Figure 6A and B). These experiments were performed following a one-hour incubation in media set to different pHext values (7.4 or 8.4) in the absence or presence of the calcium chelator, EGTA, or calcium supplementation of the media (CaCl2 , 2 mM). The one hour time point for this viability experiment was chosen for several reasons. First, we wanted to assess viability over the same time period as in the experiment measuring [Ca2+ ]i in monolayers set to different pHext values (Figure 5). Second, we noted in preliminary experiments that there was an ∼100% loss of viability when monolayers were incubated with EGTA during a 6-h alkaline stress, suggesting little possibility of reversing this severe irreversible toxicity with calcium supplementation of the media at this time point (data not shown).

% Viable cells

% Apoptotic cells

% Necrotic cells

7.4 (n = 68)

96.5

2.5

1.25

7.6 (n = 83)

95

1

4

7.8 (n = 32)

94

3

3

8.0 (n = 50)

88

10

2

8.4 (n = 60)

63

35

2

There was no evidence of apoptosis in monolayers placed in media pHext 7.4 alone in the absence or presence of calcium supplementation of the media (Figure 6A). In contrast, monolayers incubated in media pHext 7.4 containing EGTA (5 mM) demonstrated significant apoptosis, compared to control monolayers incubated in media pH7.4 alone (p < 0.001). Apoptosis with EGTA was significantly reversed with calcium supplementation (CaCl2 , 2 mM) of the media containing EGTA, compared to monolayers treated with EGTA in the absence of calcium supplementation (p < 0.01). Alkaline stress alone (pHext 8.4) for one hour induced a low level of apoptosis that was not different in the absence or presence of calcium supplementation of the media without EGTA (Figure 6B). Blockade of calcium uptake with EGTA in monolayers subjected to a similar alkaline stress for one hour amplified the development of apoptosis, compared to monolayers incubated in media/pH8.4 alone (p < 0.001) (Figure 6B). Calcium supplementation of the media in the presence of EGTA was again protective, and reduced the magnitude of apoptosis observed in media pHext 8.4 in the presence of EGTA without calcium supplementation (p < 0.01). Experiments performed using varying doses of EGTA (1, 2, 5 mM) demonstrated a dose-related increase in the percentage of apoptotic cells in both media pHext 7.4 and pHext 8.4, but the magnitude of apoptosis was significantly potentiated only at pHext 8.4 with all doses of the calcium chelator (data not shown). These experiments with blockade of calcium uptake were performed several times (n = 3) with similar results. Finally, we determined the relative change in intracellular pH induced by altering extracellular media pH. Cells were loaded with the pH-sensitive probe, BCECF, and exposed to alkaline stress for two hours in CO2 independent media (Figure 7). The relationship between extracellular pH and the fluorescent signal representing intracellular pH was linear (R-value of 0.96), demonstrating that alkalinization of the media was associated with a concomitant increase in intracellular pH in response to alkaline stress. Apoptosis · Vol 10 · No 6 · 2005

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M. Cutaia et al. Figure 3. The ultrastructural features of loss of viability following a 24-h alkaline stress assessed with transmission electron microscopy. Representative images: A: Control cells at pHext 7.4, 11,000×. B: Apoptotic cell at pHext 7.8 demonstrating dense chromatin bodies and cytoplasmic budding, 16,500×. C: Apoptotic cells at pHext 8.4 demonstrating a dense chromatin body of the largest cell 16,500×. D: Necrotic cell at pHext 7.6 demonstrating broken cell membranes and dilated endoplasmic reticulum 13,750×.

Discussion While it is well known that pH regulates many vital cell functions,1 the effect of altered pH, either extracellular or intracellular, on apoptotic signaling in endothelial cells is poorly defined. This is the first report of the induction of apoptosis in a specific endothelial cell type following alkaline stress alone, in the absence of any other cell death stimulus. We confirmed these findings using two differ1462 Apoptosis · Vol 10 · No 6 · 2005

ent methods of inducing alkaline stress, measurement of caspase activity, and both a morphological and ultrastructural assessment. Earlier work demonstrated that alkaline stress induced apoptosis in a murine fibrosarcoma line.16 As in the present work, this report demonstrated chromatin condensation following 24 h in alkaline media (pH 8.3). This work also demonstrated that an increase in extracellular pH led to a parallel increase in intracellular pH

Alkaline stress-induced apoptosis in human pulmonary artery endothelial cells Figure 4. The effect of 6-h and 24-h alkaline stress induced by CO2 -independent media on caspase-3 activity. Data represent the averaged relative fluorescent unit (RFU) ± S.E.; STSstaurosporine; n = 3 wells per condition; ∗ p < 0.01 versus media pHext 7.4 at either the 6 or 24-h incubation time, respectively.

(pHi), as noted in the present results (Figure 6), and as previously noted in response to a change in pHext.16 In fact, the increase in pHi induced by an increase in extracellular pH is variable among different cell types.23 Our prior work in HPAEC has demonstrated that in media with pHext7.4, intracellular pH is typically ∼7.1–7.2 under normal unstimulated conditions.22 The linear rela-

tionship between the relative changes in extracellular and intracellular pH noted above (Figure 6) suggests that alkaline stress induced via changes in pHext is transmitted to the intracellular compartment in HPAEC. The alterations of nuclear morphology observed with Hoechst staining, and the ultrastructural changes visualized by TEM are consistent with apoptosis. In addition, the evidence of increased caspase-3-like activity following alkaline stress demonstrates that this is a caspasedependent cell death pathway. This finding is important in light of recent work demonstrating both caspasedependent and independent forms of apoptosis,24–27 particularly in other endothelial cell types.27 Other critical signaling events in this alkaline stressinduced cell death pathway will need to be defined. Alterations in calcium ion concentration in different cellular compartments are part of the signal transduction pathway in many cellular responses, including intracellular oxidant production, altered mitochondrial function, and cell death signaling.16,28–34 Prior work in the murine fibrosarcoma line16 demonstrated that alkaline stress is associated with an increase in intracellular calcium ion concentration ([Ca2+ ]i ), but little work has been done on changes in [Ca2+ ]i in endothelial cells during alkaline stress. Prior studies in endothelial cells demonstrated different findings in regard to changes in [Ca2+ ]i in response to extracellular alkalosis, including either an

Figure 5. Measurement of intracellular calcium ([Ca2+ ]i ) using fluo-3 in HPAEC. Following loading of fluo-3, as per Methods, monolayers were incubated in media pHext 7.4 or 8.4 for 1 h, while [Ca2+ ]i was measured at baseline and at intervals for one hour; data-represent the averaged probe signal intensity (gray scale values) in all cells (n = 125) from separate monolayers (n = 5) at baseline and during the last 3-min of the measurement period in each condition; average gray scale values were obtained using the imaging software (Metamorph).

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M. Cutaia et al. Figure 6. (A and B) Blockade of calcium uptake during alkaline stress. Cells were incubated in media set to different pHext values for one hour, followed by a determination of viability, as in Figure 1; A: averaged % apoptotic nuclei ± S.E in monolayers incubated in media pHext 7.4 in the absence or presence of calcium supplementation (CaCl2 , 2 mM), or in media pHext 7.4 + EGTA (5 mM) in the absence or presence of calcium supplementation (CaCl2 , 2 mM); B: averaged % apoptotic nuclei ± S.E in monolayers incubated in media pHext 8.4 under the same conditions as in A; n ≥ 200 cells per condition, taken from four fields; ∗ p < 0.001 versus control monolayers incubated in media pH 7.4 or 8.4; ∗∗ p < 0.01 versus monolayers incubated in media pH 7.4 or 8.4 with EGTA in the absence of calcium supplementation.

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Alkaline stress-induced apoptosis in human pulmonary artery endothelial cells Figure 7. Alkaline stress induces a change in intracellular pH. Cells were incubated in media set to different pHext values for 2 h, followed by the measurement of intracellular pH (pHi) using the pH-sensitive fluoroprobe, BCECF; pHi is presented as the ratio value of 490/440 signal, as described in the Methods; R value for the regression line is 0.96.

increase or no change in [Ca2+ ]i .23,35 These prior studies documented only acute changes in [Ca2+ ]i , within minutes of a change in pHext, and were not focused on changes in viability at later time points, as in the present study. These findings prompted the experiments in the present study aimed at determining the role of altered calcium uptake in alkaline-stress induced cell death in HPAEC. An increase in [Ca2+ ]i is not a universal finding in models of apoptosis. Since both increases and decreases in [Ca2+ ]i have been observed to play a role in the initiation of apoptotic signaling in different cell types.30,33,36,37 altered calcium ion homeostasis may be a more accurate description of the role of changes in calcium uptake and [Ca2+ ]i in apoptotic signaling, as previously suggested.33 This explanation would also fit well with work suggesting that induction of apoptosis may also be linked with movement of calcium between or within intracellular compartments.32,34,38 While we did not observe a significant difference in [Ca2+ ]i during the first hour of alkaline stress at pHext 8.4, compared to monolayers incubated under normal conditions at pHext 7.4 (Figure 5), altered calcium uptake modified the magnitude of apoptosis at both pHext 7.4 and pHext 8.4 (Figure 6). Blockade of calcium uptake induced apoptosis in cells incubated in media pHext 7.4, and potentiated the magnitude of apoptosis observed in cells following alkaline stress in media pHext 8.4. The effect of calcium blockade was dose-related, and could be inhibited with calcium supplementation of the media, suggesting that altered calcium uptake is a key modulating event in the alkaline stress-induced cell death pathway in HPAEC.

The present findings in this cell death pathway in HPAEC are an extension of prior findings in which a decrease in calcium uptake, associated with a reduction in total calcium content, was linked to the initiation of apoptosis in several other cell types.33,36,37 Most of this prior work was performed using malignant cell lines. Additional experiments, beyond the scope of the present work, will be required to further define the alterations of calcium ion homeostasis in the alkaline stress model in HPAEC, and to determine if blockade of calcium uptake is a generalized signaling event in this model in other normal cell lines. Changes in both extracellular and intracellular pH are important stimuli for the initiation of physiologically relevant signal transduction.1,23,39–41 We have previously demonstrated an increase in endogenous oxidant production in HPAEC when intracellular pH is increased during a severe metabolic insult.40 Furthermore, changes in pH and calcium homeostasis in cells are often closely linked, especially in cell death pathways.16,42,43 The potentiation of alkaline stress-induced cell death with blockade of calcium uptake suggests that a pH-hydrogen ion/calcium signaling interaction modifies the magnitude of apoptosis in the alkaline stress-induced cell death in HPAEC. Thus, we postulate that one or more calcium signaling events may be critical regulators of the magnitude of cell death under these conditions. Apoptosis is being reported with increasing frequency in conditions associated with organ-system injury.44–46 The present findings support and extend recent work demonstrating that alkalosis may potentiate injury in the lung at the organ-system level.13,17 Additional work will be required to completely define the components of the alkaline stress-induced apoptotic pathway in lung endothelium, and the potential clinical benefit of modifying this pathway to preserve endothelial cell viability in vivo.

Acknowledgments

This work was supported by a Veterans Administration Merit Review Grant to M. Cutaia.

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