Osmotically induced cytosolic free Ca 2+ changes in human neutrophils

Abstract

Cytosolic free Ca 2+ concentration in neutrophils was measured by ratiometric fluorometry of intracellular fura2. Increasing the extracellular osmolarity, by either NaCl (300–600 mM) or sucrose (600–1200 mM), caused a rise in cytosolic free Ca 2+ (Δ max≅600 nM). This was not due to cell lysis as the cytosolic free Ca 2+ concentration was reversed by restoration of isotonicity and a second rise in cytosolic free Ca 2+ could be provoked by repeating the change in extracellular osmolarity. Furthermore, the rise in cytosolic free Ca 2+ concentration occurred in the absence of extracellular Ca 2+, demonstrating that release of intracellular fura2 into the external medium did not occur. The osmotically-induced rise in cytosolic free Ca 2+ was not inhibited by either the phospholipase C-inhibitor U73122, or the microfilament inhibitor cytochalasin B, suggesting that neither signalling via inositol tris-phosphate or the cytoskeletal system were involved. However, the rise in cytosolic free Ca 2+ may have resulted from a reduction in neutrophil water volume in hyperosmotic conditions. As these rises in cytosolic Ca 2+ (Δ max≅600 nM) were large enough to provoke changes in neutrophil activity, we propose that conditions which removes cell water may similarly elevate cytosolic free Ca 2+ to physiologically important levels.