Abstract
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. In conclusion: 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.
Highlights
From the Infectious Diseases Division and the JFondation pourla Recherche Mkdicale, University Hospital, 1211 Genkve 4, Switzerland and the Slnstitut fur Physiologische Chemie, Ruhr Universitat Bochum,0-4630 Bochum, Federal Republic of Germany
We have recently demonstrated that the sustained rise in [Ca2+Iin human neutrophils is related to Ca2+influx, as shown from itsinhibition by polyvalent metal ions (9)
Cb fMLP Stimulation of Ca2+Influx Assessed by Quenchingof Quin[2] or Fura-2 Fluorescence by Mn2+-Mn2+ quenches intracellular quin[2] or fura-2 fluorescence effectively (23, 24), and this property was initially exploited for the purpose of calibration (24, 25).More recently, it has been shown by fluorimetric studies based on quenching that agonists can promote Mn2+influx across the plasma membrane (9, 10)
Summary
From the Infectious Diseases Division and the JFondation pourla Recherche Mkdicale, University Hospital, 1211 Genkve 4, Switzerland and the Slnstitut fur Physiologische Chemie, Ruhr Universitat Bochum,0-4630 Bochum, Federal Republic of Germany. It was part of this study to cells, respectively), a progressive reduction of both[CaZ+li characterize the interrelation between these reactions, an important consideration in thiscontext being whether initial tion, namely Ins(1,4,5)P3, Ins(1,3,4)P3, and Ins(1,3,4,5)Pd, signaling events, i.e. Ca2+ mobilization, activation of Ca2+ were analyzed in HL-60 cells 1 min after stimulation with influx, and generation of inositol phosphates, require the same increasing doses of fMLP (Fig. 9, lowerpanel).Note that Ca2+
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
