Staurosporine clamps cytosolic free Ca2+ concentrations of human neutrophils.
The present studies indicate that 50 nM-10 microM-staurosporine increased cytosolic free Ca2+ concentrations ([Ca2+]i) of fura-2-loaded neutrophils in a non-linear manner. The rise in [Ca2+]i was rapid, reaching a plateau (e.g. to 0.4 microM with 1 microM-staurosporine) within 30 s, and was maintained for more than 20 min. Pretreating cells with pertussis toxin had no effect on this reaction. The elevation of [Ca2+]i was insensitive to extracellular Ca2+ concentrations and was due entirely to mobilization of intracellular Ca2+ stores. Mn(2+)-quench studies confirmed the absence of Ca2+ influx. No Ca2+ efflux occurred in staurosporine-treated cells. In combination studies, staurosporine potentiated Ca2+ influx induced by N-formylmethionyl-leucyl-phenylalanine (FMLP) and did not block Ca2+ efflux associated with peptide stimulation of neutrophils. Studies with permeabilized cells showed that staurosporine did not directly release intracellular Ca2+ stores, nor did it affect the sequestration of Ca2+ by a Ca2+/ATPase pump. A radioligand-binding assay failed to detect changes in the level of inositol 1,4,5-trisphosphate in neutrophils incubated with less than or equal to 1 microM-staurosporine, but in cells treated with 10 microM-staurosporine the assay recorded a transient increase in this second messenger similar to that induced by FMLP. Finally, lysozyme, but not beta-glucuronidase, was released from staurosporine-treated cells. The present results suggest that staurosporine increased [Ca2+]i by indirectly mobilizing internal Ca2+ stores. Staurosporine suppression of Ca2+ efflux and generation of a persistent signal may account for the maintained elevation of [Ca2+]i.
Wong
K
K
Department of Medicine and Pharmacology, University of Alberta, Edmonton, Canada.
Kwan-Yeung
L
L
Turkson
J
J
D000470:Alkaloids / Q000494:pharmacology*; D002118:Calcium / Q000097:blood*; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D016257:Fura-2; D005966:Glucuronidase / Q000097:blood; D006801:Humans; D066298:In Vitro Techniques; D015544:Inositol 1,4,5-Trisphosphate / Q000097:blood; D015759:Ionomycin / Q000494:pharmacology; D007700:Kinetics; D008247:Lysosomes / Q000201:enzymology; D008345:Manganese / Q000494:pharmacology; D009113:Muramidase / Q000097:blood; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D037342:Pertussis Toxin; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*; D013050:Spectrometry, Fluorescence; D019311:Staurosporine; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D000470:Alkaloids; D010566:Virulence Factors, Bordetella; D008345:Manganese; D015759:Ionomycin; D009240:N-Formylmethionine Leucyl-Phenylalanine; D015544:Inositol 1,4,5-Trisphosphate; D037342:Pertussis Toxin; D011493:Protein Kinase C; D009113:Muramidase; D005966:Glucuronidase; D019311:Staurosporine; D002118:Calcium; D016257:Fura-2