Ca2+ transients and Mn2+ entry in human neutrophils induced by thapsigargin.
Human neutrophils, preloaded with the fluorescent probe, Fura-2, were exposed to Ca2+-releasing agents. The monitored traces of fluorescence were transformed by computer to cytosolic Ca2+ concentration ([ Ca2+]i). Due to quenching of Fura-2, the addition of Mn2+ enabled us to compute the cytosolic concentration of total manganese ([Mn]i). The agents used were the novel Ca2+-mobilizing agent, thapsigargin (Tg), the chemotactic peptide, formyl-methionyl-leucyl-phenylalanine (FMLP), and the divalent cation ionophore, A23187. The agents caused transient rises of [Ca2+]i and monotonous rises of [Mn]i, suggesting influx but no efflux of Mn2+. The rise time of [Ca2+]i and the time constants and magnitude of the apparent Mn2+ influx were strongly dependent on the sequence of addition of the agonist and Ca2+. Contrary to FMLP, Tg needed several minutes to exert its full effect on the rise of [Ca2+]i and on the influx of Mn2+, the latter being dependent on two phases, activation and partial inactivation. Pretreatment with phorbol 12-myristate 13-acetate (PMA) inhibited the responses of Tg, FMLP and A23187. For comparison, human red blood cells were tested. Contrary to A23187, Tg did not induce Ca2+ uptake in ATP-depleted red cells but increased the Ca2+ pump flux in intact red cells by 10%. The experimental data and computer simulations of the granulocyte data suggest that time-dependent changes of both passive Ca2+ flux into the cytosol and Ca2+ flux of the plasma membrane pump are involved in the transient [Ca2+]i response.
Foder
B
B
Department of Clinical Physiology and Nuclear Medicine University Hospital, Copenhagen, Denmark.
Scharff
O
O
Thastrup
O
O
D001572:Benzofurans; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D003198:Computer Simulation; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism; D005456:Fluorescent Dyes; D016257:Fura-2; D006706:Homeostasis; D006801:Humans; D008345:Manganese / Q000378:metabolism*; D008433:Mathematics; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D010936:Plant Extracts / Q000494:pharmacology*; D013729:Terpenes / Q000494:pharmacology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D019284:Thapsigargin; D013997:Time Factors
D001572:Benzofurans; D005456:Fluorescent Dyes; D010936:Plant Extracts; D013729:Terpenes; D000001:Calcimycin; D008345:Manganese; D009240:N-Formylmethionine Leucyl-Phenylalanine; D019284:Thapsigargin; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; D016257:Fura-2