Abstract
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.
Similar content being viewed by others
References
Busa WB, Nuccitelli R. Metabolic regulation via intracellular pH. Am J Physiol (Regulatory Integrative Comp Physiol) 1984; 246: R409–R438.
Gillies RJ, Martinez-Zaguilan R, Martinez GM, Serrano R, Perona R. Tumorigenic 3T3 cells maintain an alkaline intracellular pH under physiological conditions. Proc Natl Acad Sci 1990; 87: 7414–7418.
Vairo G, Cocks BG, Cragoe EJ, Hamilton JA. Selective suppression of growth factor-induced cell cycle gene expression by Na/H antiport inhibitors. J Biol Chem 1992; 267: 19043–19046.
Takeshita K, Suzuki Y, Nishio K, et al. Hypercapnic acidosis attenuates endotoxin-induced nuclear factor κB activation. Am J Respir Cell Mol Biol 2003; 29: 124–132.
Mizuno S, Demura Y, Ameshima S, Okamura S, Miyamora I, Ishizaki Y. Alkalosis stimulates endothelial nitric oxide synthase in cultured human pulmonary artery endothelial cells. Am J Physiol Lung Cell Mol Physiol 2002; 283: L113–L119.
Xu L, Fidler IJ. Acidic pH-induced elevation in interleukin-8 expression by human ovarian carcinoma cells. Cancer Res 2000; 60: 4610–4616.
Barry MA, Reynolds JE, Eastmann A. Etoposide induced apoptosis in human HL60 cells is associated with intracellular acidification. Cancer Res 1993; 53: 2349–2357.
Wolf CM, Eastman A. Intracellular acidification during apoptosis can occur in the absence of a nucleus. Biochemical & Biophysical Res Comm 1999; 254: 821–827.
Li J, Eastman A. Apoptosis in a Interleukin-2-dependent cytotoxic T lymphocyte cell line is associated with intracellular acidification, role of the Na+/H+ antiport. J Biol Chem 1995; 270: 3203–3211.
Rebollo A, Gomez J, Martinez A, Lastres P, Silva A, DP-S. Apoptosis induced by IL-2 withdrawel is associated with intracellular acidification. Experimental Cell Research 1995; 218: 581–585.
Terminella CK, Tollefson K, Kroczynski J, Pelli J, Cutaia M. Inhibition of apoptosis in pulmonary endothelial cells by altered pH, mitochondrial function, and ATP supply. Am J Physiol (Lung Cell Mol Physiol) 2002; 283: L1291–L1297.
Dai HY, Tsao N, Leung WC, Lei HY. Increase in intracellular pH in p53-dependent apoptosis of thymocytes induced by gamma radiation. Radiat Res 1998; 150: 183–189.
Laffey JG, Tanaka M, Engelberts D, et al. Therapeutic hypercapnia reduces pulmonary and systemic injury following in vivo lung reperfusion. Am J Respir Crit Care Med 2000; 162: 2287–2294.
Cutaia M, Tollefson K, Kroczynski J, Parks N, Rounds S. Role of the Na/H antiport in pH-dependent cell death in pulmonary artery endothelial cells. Am J Physiol (Lung Cell Mol Physiol) 2000; 278: L536–L544.
Lemasters JJ. Mechanisms of hepatic toxicity: Necrapoptosis and the mitochondrial permeability transition: Shared pathways to necrosis and apoptosis. Am J Physiol (Gastrointest Liver Physiol) 1999; 276: G1–G6.
Majima HJ, Oberley TD, Furukawa K, et al. Prevention of mitochondrial injury by manganese superoxide dismutase reveals a primary mechanism for alkaline-induced cell death. J Biol Chem 1998; 273: 8217–8224.
Laffey JG, Engelberts D, Kavanagh BP. Injurious effects of hypocapnic alkalosis in the isolated lung. Am J Respir Crit Care Med 2000; 162: 399–405.
Tsao N, Lei HY. Activation of the Na/H antiporter, Na/HCO3−/CO32− cotransporter, or Cl−/HCO3− exchanger in spontaneous thymocyte apoptosis. The Journal of Immunology 1996; 157: 1107–1116.
Khaled AR, Moor AN, Li A, et al. Trophic factor withdrawal: p38 mitogen activated protein kinase activates NHE1 which induces intracellular alkalinization. Mol Cell Biol 2001; 21: 7545–7557.
Ziegelstein RC, Cheng L, Blank PS, et al. Modulation of calcium homeostasis in cultured rat aortic endothelial cells by intracellular acidification. Am J Physiol (Heart Circ Physiol) 1995; 265: H1424–H1433.
Majno G, Joris I. Apoptosis, oncosis, and necrosis: An overview of cell death. Am J Pathology 1995; 146: 3–15.
Cutaia M, Parks N. Effect of hyperoxia and exogenous oxidant stress on pulmonary artery endothelial cell Na/H antiport activity. J Lab Clin Med 1996; 128: 154–164.
Wakabayashi I, Groschner K. Divergent effects of extracellular and intracellular alkalosis on Ca2+ entry pathways in vascular endothelial cells. Biochem J 1997; 323: 567–573.
Daugas E, Susin SA, Zanzami N, et al. Mitochondrio-nuclear translocation of AIF in apoptosis and necrosis. FASEB J 2000; 14: 729–739.
Cande C, Cecconi F, Dessen P, Kroemer G. Apoptosis-inducing factor (AIF): Key to the conserved caspase-independent pathways of cell death. J Cell Sci 2002; 115: 4727–4734.
Nur-E-Kamal A, Gross SR, Pan Z, Balklava Z, Ma J, Liu LF. Nuclear translocation of cytochrome c during apoptosis. J Biol Chem 2004; 279: 24911–24914.
Zhang W, Li D, Mehta JL. Role of AIF in human coronary artery endothelial cell apoptosis. Am J Physiol 2004; 286: H354–H358.
Berridge MJ, Bootman MD, Lipp P. Calcium-a life and death signal. Nature 1998; 395: 645–648.
Schild L, Keilhoff G, Augustin W, Reiser G, Striggow F. Distinct Ca2+ thresholds determine cytochrome c release or permeability transition pore opening in brain mitochondria. FASEB J 2001; 15: 565–567.
Mussche S, Leybaert L, D'Herde K. First and second role of calcium: Survival versus apoptosis in serum-free cultured granulosa explants. Annals New York Acad Sci 2000; 926: 101–115
Qian T, Herman B, Lemasters JJ. The mitochondrial permeability transition mediates both necrotic and apoptotic cell death of hepatocytes exposed to Br-A23187. Toxicology and Applied Pharmacology 1999; 154: 117–125.
Kruman II, Mattson MP. Pivotal role of mitochondrial calcium uptake in neural cell apoptosis and necrosis. J Neurochem 1999; 72: 529–540.
Kluck RM, McDougall CA, Harmon BV, Halliday JW. Calcium chelators induce apoptosis-evidence that raised intracellular ionised calcium is not essential for apoptosis. Biochim Biophys Acta 1994; 1223: 247–254.
Cerella C, D'Alessio M, De Nicola M, Magrini A, Bergamaschi A, Ghibelli L. Cytosolic and endoplasmic reticulum Ca2+ concentrations determine the extent and the morphological type of apoptosis, respectively. Ann NY Acad Sci 2003; 1010: 74–77.
Nishio K, Suzuki Y, Takeshita K, et al. Effects of hypercapnia and hypocapnia on [Ca2+] i mobilization in human pulmonary artery endothelial cells. J Appl Physiol 2001; 90: 2094–2100.
Baffy G, Miyashita T, Williamson JR, Reed JC. Apoptosi induced by withdrawal of Interleukin-3 (IL-3) from an IL-3 dependent hematopoietic cell line is associated with repartitioning of intracellular calcium and is blocked by enforced Bcl-2 oncoprotein production. J Biol Chem 1993; 268: 6511–6519.
Bansal N, Houle AG, Melnykovych G. Dexamethasone-induced killing of neoplastic cells of lymphoid derivation: Lack of early calcium involvement. J Cell Physiol 1990; 143: 105–109.
Zhu WH, Loh TT. Roles of calcium in the regulation of apoptosis in HL-60 promyelocytic leukemia cells. Life Sci 1995; 57: 2091–2099.
Marumo M, Suehiro A, Kakishita E, Groschner K, Wakabayashi I. Extracellular pH effects platelet aggregation associated with modulation of store-operated Ca2+ entry. Thromb Res 2001; 104: 353–360.
Cutaia M, Kroczynski J, Tollefson K. pH-dependent oxidant production following inhibition of the mitochondrial electron transport chain in pulmonary endothelial cells. Endothelium 2002; 9: 109–121.
Shrode LD, Tapper H, Grinstein S. Role of intracellular pH in proliferation, transformation, and apoptosis. J Bioenerg Biomembr 1997; 29: 393–399.
Trump BF, Berezesky IK. Calcium-mediated cell injury and cell death. FASEB J 1995; 9: 219–228.
Silverman HS, Stern MD. Ionic basis of ischaemic cardiac injury: Insights from cellular studies. Cardiovascular Research 1994; 28: 581–597.
Narula J, Pandey P, Arbustini E, et al. Apoptosis in heart failure: Release of cytochrome c from mitochondria and activation of caspase-3 in human cardiomyopathy. Proc Natl Acad Sci 1999; 96: 8144–8149.
Fischer S, Maclean AA, Liu M, et al. Dynamic changes in apoptotic and necrotic cell death correlate with severity of ischemia-reperfusion injury in lung transplantation. Am J Respir Crit Care Med 2000; 162: 1932–1939.
Kawasaki M, Kuwano K, Hagimoto N, et al. Protection from lethal apoptosis in lipopolysaccharide-induced acute lung injury in mice by a caspase inhibitor. Am J Pathol 2000; 157: 597–603.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Cutaia, M., Black, A.D., Cohen, I. et al. Alkaline stress-induced apoptosis in human pulmonary artery endothelial cells. Apoptosis 10, 1457–1467 (2005). https://doi.org/10.1007/s10495-005-1402-5
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10495-005-1402-5