Chemoattractant receptor promotion of Ca2+ influx across the plasma membrane of HL-60 cells. A role for cytosolic free calcium elevations and inositol 1,3,4,5-tetrakisphosphate production.
The mechanisms by which the chemotactic peptide formyl-methyl-leucyl-phenyl-alanine stimulates Ca2+ influx across the plasma membrane were investigated in the human promyelocytic cell line HL-60, induced to differentiate with dimethyl sulfoxide. Ca2+ influx was determined: (a) from the initial rate of Mn2+ influx, apparent from the quenching of intracellular quin2 or fura-2 fluorescence; (b) from the rate of the elevation of cytosolic free calcium, [Ca2+]i, upon readdition of Ca2+ to cells previously stimulated in the absence of extracellular Ca2+. [3H]Inositol tris-, tetrakis-, and pentakisphosphates were analyzed by a high performance liquid chromatography procedure which was optimized for the separation of inositol tetrakisphosphates, yielding three predominant isomers: inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4), inositol 1,4,5,6-tetrakisphosphate, and inositol 1,3,4, 6-tetrakisphosphate. Both the kinetics and agonist dose dependence of Ca2+ influx stimulation correlated closely with the corresponding receptor-mediated variations of [Ca2+]i either in the presence or in the absence of extracellular Ca2+. Of the different inositol phosphates determined in parallel and under the same conditions, accumulation of [3H]Ins(1,3,4,5)P4 correlated best with Ca2+ influx both temporally and in its dose dependence in the presence or in the absence of extracellular Ca2+; inositol 1,3,4-trisphosphate was also correlated but to a lesser extent. Attenuations of [Ca2+]i elevations by decreasing extracellular Ca2+ or by increasing the cytosolic Ca2+ buffering capacity with quin2 led to parallel inhibition of Ca2+ influx and Ins(1,3,4,5)P4 production.


CONCLUSIONS
1) activation of Ca2+ influx by formyl-methionyl-leucyl-phenylalanine depends on the elevation of [Ca2+]i, the latter being initiated by Ca2+ mobilization from intracellular stores; 2) Ins(1,3, 4,5)P4 is a strong candidate for maintaining receptor-mediated activation of Ca2+ influx in differentiated HL-60 cells.
Pittet
D
D
Infectious Diseases Division, University Hospital, Genève, Switzerland.
Lew
D P
DP
Mayr
G W
GW
Monod
A
A
Schlegel
W
W
D000634:Aminoquinolines / Q000494:pharmacology; D001692:Biological Transport; D002118:Calcium / Q000502:physiology*; D015220:Calcium Channels / Q000502:physiology; D002462:Cell Membrane / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007295:Inositol Phosphates / Q000502:physiology*; D007536:Isomerism; D007700:Kinetics; D015473:Leukemia, Promyelocytic, Acute; D008856:Microscopy, Fluorescence; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D009504:Neutrophils / Q000502:physiology*; D013403:Sugar Phosphates / Q000502:physiology*; D014407:Tumor Cells, Cultured
D000634:Aminoquinolines; D015220:Calcium Channels; D007295:Inositol Phosphates; D013403:Sugar Phosphates; D009240:N-Formylmethionine Leucyl-Phenylalanine; C035738:Quin2-acetoxymethyl ester; D002118:Calcium