Abstract
Stimulation of human neutrophils with f-met-leu-phe, platelet-activating factor, or leukotriene B4 resulted in an increase in [Ca2+]i. The [Ca2+]i rise was greater in the presence than absence of external Ca2+; the component that was dependent on external Ca2+ was blocked by Ni2+, or could be reconstituted by addition of external Ca2+ following discharge of the internal Ca2+ store. These measurements of [Ca2+]i responses provide only indirect evidence for agonist-stimulated Ca2+ entry, and here we have used an alternative approach to demonstrate directly agonist-stimulated divalent cation entry. In the presence of extracellular Mn2+, f-met-leu-phe, leukotriene B4, and platelet-activating factor stimulate a quench in fluorescence of fura-2-loaded human neutrophils. This quench was due to stimulated Mn2+ influx and was blocked by Ni2+. When Mn2+ was added in the continued presence of agonist, after discharge of the internal store of Ca2+, a stimulated quench was seen; this result shows that an elevated [Ca2+]i is not needed for the stimulation of Mn2+ entry. Depolarization by high [K+] or addition of the L-type Ca2+ channel agonist, BAY-R-5417, had little or no effect on either [Ca2+]i or Mn2+ entry. These results show that agonists stimulate divalent cation entry (Ca2+ or Mn2+) by a mechanism independent of changes in [Ca2+]i and unrelated to voltage-dependent Ca2+ channels.
Highlights
Stimulation ofhuman neutrophils withf-met-leu- ments [1,2,3,4,5,6] have been carried out using quin2-loaded neutrophe, platelet-activating factor, or leukotriene B4 re- phils where stimulation causes significant changes in autoflusulted in an increase in [Ca2+]i.The [Ca2+Iirise was orescence and the amounts of greater in the presence than absence of external Ca2+; quin2 required to obtaina sufficient signal tend to buffer the component that was dependent on external Ca2+ changes in [Ca2+Ii[1].the use of intracellular was blocked by Ni2+,or could be reconstituted by addition of external Ca2+following discharge of the internal Ca2+store
Platelet-activating factor stimulate a quench in fluo- stimulated Ca2+influx [8].Here, we have used an alternative rescence of fura-2-loaded human neutrophils
The shape and extent of the rise seems to be dependent on the agonist and resembles the response seen in the continued presence of external Ca2+.The response to the addition of Ca2+in the presence of LTB4was considerably smaller than that seen with PAF or fMLP andcorresponds to thesmaller
Summary
Q 1989 by The American Society for Biochemistry and Molecular Biology, Inc. Vol 264, No 3, Issue of January 25, pp. 1522-1527,1989 Printed in U.S.A. Stimulation ofhuman neutrophils withf-met-leu- ments [1,2,3,4,5,6] have been carried out using quin2-loaded neutrophe, platelet-activating factor, or leukotriene B4 re- phils where stimulation causes significant changes in autoflusulted in an increase in [Ca2+]i.The [Ca2+Iirise was orescence (due to NADPH oxidation) and the amounts of greater in the presence than absence of external Ca2+; quin required to obtaina sufficient signal tend to buffer the component that was dependent on external Ca2+ changes in [Ca2+Ii[1].the use of intracellular was blocked by Ni2+,or could be reconstituted by addition of external Ca2+following discharge of the internal Ca2+store These measurements of [Ca2+]i responses provide only indirect evidence for agoniststimulated Ca2+ entry, and here we have used an alternative approach to demonstrate directly agonistdyes to measure [Ca2+Iiprovides only indirect evidence for a stimulated Ca2+influx. All the records are typical of at leastthreeothers obtained from different cell preparations
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