Osmotic forces are not critical for Ca2+-induced secretion from permeabilized human neutrophils.
In order to examine the role of osmotic forces in degranulation, the effects of solutes and osmolality on granule secretion were explored using both FMLP-stimulated, intact neutrophils and Ca2+-stimulated, permeabilized cells. We employed a HEPES-based buffer system which was supplemented with: a) permeant (KCl or NaCl) or impermeant (Na-isethionate or choline-Cl) ions, or b) permeant (urea) or impermeant (sucrose) uncharged solutes. Intact and permeabilized cells had significantly different solute requirements for degranulation. FMLP-stimulated release from intact cells was supported by NaCl or Na-isethionate greater than KCl greater than choline-Cl or sucrose greater than urea. In contrast, the rank order of Ca2+-stimulated release from permeabilized cells was choline-Cl greater than Na-isethionate, KCl, or NaCl greater than sucrose greater than urea. Hypo-osmotic conditions caused increased levels of background granule release from both intact and permeabilized neutrophils. However, hypo-osmolality inhibited both FMLP-stimulated degranulation from intact cells and Ca2+-induced release from permeabilized neutrophils. While hyperosmotic conditions inhibited stimulated release from intact cells, this inhibition was much less pronounced in permeabilized cells when the granules were directly exposed to these solutions. In fact, hyperosmotic sucrose greatly enhanced Ca2+-induced secretion. Although isolated specific and azurophil granules showed some lytic tendencies in hypo-osmotic buffers, the overall stability of the isolated granules did not indicate that swelling alone could effect degranulation. These results suggest that degranulation in permeabilized cells is neither due to nor driven by simple osmotic forces (under resting or stimulated conditions) and emphasize differences obtained by bathing both the granules and plasma membrane (as opposed to membranes alone) in various solutes.
Stoehr
S J
SJ
Department of Pediatric Hematology-Oncology, University of Michigan Medical Center, Ann Arbor 48109.
Smolen
J E
JE
D002118:Calcium / Q000494:pharmacology*; D002463:Cell Membrane Permeability; D003594:Cytoplasmic Granules / Q000187:drug effects / Q000502:physiology; D004072:Digitonin / Q000494:pharmacology; D005966:Glucuronidase / Q000097:blood; D006801:Humans; D007700:Kinetics; D009113:Muramidase / Q000097:blood; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000502:physiology*; D009994:Osmolar Concentration; D014155:Transcobalamins / Q000032:analysis
D014155:Transcobalamins; D009240:N-Formylmethionine Leucyl-Phenylalanine; D009113:Muramidase; D005966:Glucuronidase; D004072:Digitonin; D002118:Calcium