title: Micromolar concentrations of free calcium provoke secretion of lysozyme from human neutrophils permeabilized with saponin.
abstract: Permeabilization of human neutrophils has been accomplished by using saponin, a cholesterol complexing agent, permitting experimental manipulation of the intracellular milieu. Access of ordinarily impermeable solutes, such as [14C]-inulin or [14C]-sucrose, to the water space of the cells was considered the main criterion for permeabilization. Other criteria were substantial (50 to 80%) release of cytoplasmic lactate dehydrogenase and permeability to trypan blue. Successful permeabilization did not cause substantial release of the granule enzymes lysozyme or beta-glucuronidase. Washing the neutrophils, to remove soluble saponin and released cytoplasmic contents, and resuspension did not alter their permeabilized character. By supplementing the medium with CaCl2, thereby obtaining free Ca2+ concentrations of 1.5 X 10(-7) M to 10(-4) M, it was possible to stimulate lysozyme secretion from washed or unwashed permeabilized neutrophils. A total of 20 to 30% of the total cellular lysozyme was released during an incubation of 5 min at 37 degrees C. Secretion was inversely related to cell concentration. No beta-glucuronidase was secreted under these conditions and no response was obtained by using unpermeabilized cells. Thus, permeabilized neutrophils respond to increases in free Ca2+ alone, without resorting to conventional secretagogues. This system also permits the manipulation of intracellular constituents important for stimulus-response coupling.
mesh_terms: D000328:Adult; D002021:Buffers; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D002463:Cell Membrane Permeability / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D009113:Muramidase / Q000378:metabolism*; D009504:Neutrophils / Q000201:enzymology* / Q000378:metabolism; D012503:Saponins; D013449:Sulfonamides / Q000494:pharmacology; D014268:Trifluoperazine / Q000494:pharmacology
chemical_list: D002021:Buffers; D012503:Saponins; D013449:Sulfonamides; D014268:Trifluoperazine; C017967:W 7; D007770:L-Lactate Dehydrogenase; D009113:Muramidase; D002118:Calcium