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Lab Invest. 1989 May;60(5):630-42.

Cytosolic ionized calcium and bleb formation after acute cell injury of cultured rabbit renal tubule cells.

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1
Department of Pathology, University of Maryland School of Medicine, Baltimore.

Abstract

Changes in cytosolic calcium ([Ca2+]i) and cell blebbing of cultured rabbit kidney proximal tubule cells were studied in response to injury induced through a variety of mechanisms. [Ca2+]i was measured in Fura 2-loaded cells and blebbing was observed by phase microscopy. The severity of injury was evaluated by electron microscopy and cell killing was estimated by trypan blue dye uptake. The types of injury included interaction with sulfhydryl groups (HgCl2, N-ethylmaleimide, p-chloromercuribenzene sulfonic acid, inhibition of energy metabolism (carbonyl cyanide m-chlorophenylhydrazone, KCN, KCN + iodoacetate) and ion deregulation (ouabain, ionomycin, A23187). The role of extracellular calcium ([Ca2+]e) in injury was also studied. HgCl2, N-ethylmaleimide and ionomycin + [Ca2+]e caused the highest elevations of [Ca2+]i, the most extensive blebbing, and most rapid cell death. P-chloromercuribenzene sulfonic acid treatment resulted in a moderate increase in [Ca2+]i, as well as less extensive blebbing and slower cell death. Ouabain and inhibitors of mitochondrial and cellular energy metabolism caused only a 2-fold increase in [Ca2+]i, a few blebs and delayed cell death. Ionomycin - [Ca2+]e caused a transient elevation of [Ca2+]i, minimal blebbing and very slow cell killing. The increase in [Ca2+]i may result from redistribution of intracellular stores (N-ethylmaleimide, p-chloromercuribenzene sulfonic acid, KCN, carbonyl cyanide m-chlorophenylhydrazone, ionomycin - [Ca2+]e), from influx of extracellular [Ca2+]e (ionomycin + [Ca2+]e, ouabain), or from both redistribution and influx (HgCl2). Therefore, removing [Ca2+]e is protective only in certain types of injury, (HgCl2, ionomycin). Cytoplasmic blebbing was seen with all the types of injury studied and occurred before to cell death. Blebs formed rapidly, enlarged, and sometimes detached with membrane sealing. Our results indicate that cell injury which initiates a 3-fold or greater sustained elevation in [Ca2+]i, resulting from either an influx of [Ca2+]e or by Ca2+ release from intracellular pools, is also associated with abundant bleb formation and rapid cell death.

PMID:
2654473
[Indexed for MEDLINE]

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