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Rapid kinetics of agonist-evoked changes in cytosolic free Ca2+ concentration in fura-2-loaded human neutrophils.

ABSTRACT: The initial kinetics of agonist-evoked rises in the cytosolic Ca2+ concentration [Ca2+]i were investigated in fura-2-loaded human neutrophils by stopped-flow fluorimetry. The rises in [Ca2+]i evoked by chemotactic peptide (fMet-Leu-Phe), platelet-activating factor and ADP all lagged behind agonist addition by 1-1.3 s. Lag times were not significantly different in the presence and in the absence of external Ca2+. Stimulation of the cells in the presence of extracellular Mn2+ resulted in a quench of fluorescence with a similar lag time to [Ca2+]i rise. The delay in onset of the rise in [Ca2+]i evoked by fMet-Leu-Phe was dependent on concentration, becoming longer at lower concentrations of agonist. These results indicate that both the agonist-evoked discharge of the intracellular Ca2+ stores and the generation of bivalent-cation influx lag behind agonist-receptor binding in neutrophils. Both pathways thus appear to be mediated by indirect mechanisms, rather than by a directly coupled process such as a receptor-operated channel. The temporal coincidence of the onset of store discharge with the commencement of bivalent-cation influx suggests that the two events may be causally linked.

SUBMITTER: Sage SO

PROVIDER: S-EPMC1133721 | biostudies-other | 1990 Feb

REPOSITORIES: biostudies-other

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Spiking in cytosolic calcium concentration in single fibrinogen-bound fura-2-loaded human platelets.

Project description:Fura-2-loaded human platelets were immobilized on a fibrinogen-coated surface and the cytosolic free calcium concentration ([Ca2+]i) was measured in single platelets by low-light-level video-ratio image-processing of the optical probe signal. Some fibrinogen-bound platelets showed repetitive spiking in [Ca2+]i with a mean frequency of about 2/min, which increased to 5/min in the presence of ADP. Other cells showed no activity until the addition of agonist. When immobilized in the presence of prostaglandin I2 and the fibrinogen antagonist Arg-Gly-Asp-Ser, the platelets adhered less firmly to fibrinogen, and in many [Ca2+]i remained low and constant. Subsequent activation of such platelets with ADP evoked oscillations in [Ca2+]i with a peak frequency of about 5/min and which persisted for at least 5 min. These results indicate that human platelets, like many other non-excitable cells, have an elaborate system of calcium signalling involving spiking.

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| S-EPMC1137488 | biostudies-other

Activation of protein kinase C in human neutrophils attenuates agonist-stimulated rises in cytosolic free Ca2+ concentration by inhibiting bivalent-cation influx and intracellular Ca2+ release in addition to stimulating Ca2+ efflux.

Project description:Stimulation of fura-2-loaded human neutrophils with formylmethionyl-leucyl-phenylalanine (FMLP) or ionomycin elevated the cytosolic free Ca2+ concentration, [Ca2+], to a maintained elevated level. Activation of protein kinase C (C-kinase) with phorbol 12-myristate 13-acetate, 4 beta-phorbol 12,13-didecanoate or dioctanoylglycerol caused decreases in [Ca2+]i from this level. 4 alpha-Phorbol didecanoate, which does not activate C-kinase, had no effect. These results confirm previous reports that C-kinase activation decreases neutrophil [Ca2+]i by stimulating removal of Ca2+ from the cytosol. Further experiments showed that activation of C-kinase attenuated the component of the FMLP-stimulated [Ca2+]i rise that was dependent on external Ca2+. C-kinase activation also inhibited FMLP-stimulated entry of the quenching cation, Mn2+, used as an indicator of bivalent-cation entry. In contrast, C-kinase activation caused only a partial inhibition of FMLP-stimulated release of Ca2+ from intracellular stores. 4 alpha-Phorbol didecanoate was ineffective in inhibiting Ca2+ entry, Mn2+ entry and intracellular Ca2+ release. Addition of FMLP also stimulated a decrease in the ionomycin-elevated [Ca2+]i, and this effect was blocked by staurosporine, a protein kinase inhibitor. These results show that, in addition to stimulating Ca2+ efflux, C-kinase activation in neutrophils inhibits FMLP-stimulated entry of bivalent cations, and partially inhibits intracellular release of Ca2+. Further, FMLP itself can modulate [Ca2+]i by activation of C-kinase.

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ADP evokes biphasic Ca2+ influx in fura-2-loaded human platelets. Evidence for Ca2+ entry regulated by the intracellular Ca2+ store.

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Project description: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.

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