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Stimulus-response coupling in human platelets. Changes evoked by platelet-activating factor in cytoplasmic free calcium monitored with the fluorescent calcium indicator quin2.

ABSTRACT: The role of changes in cytoplasmic free calcium, [Ca2+]i, in the responses to platelet-activating factor (PAF) was studied in human platelets loaded with the fluorescent calcium indicator, quin2. In the presence of 1 mM external calcium, PAF raised [Ca2+]i 8-10-fold in a few seconds to peak near 1 microM. [Ca2+]i then declined over several minutes towards the basal level. In the absence of external calcium there was a much smaller increase in [Ca2+]i of similar pattern. These findings suggest that PAF increases [Ca2+]i partly by discharge of internal Ca2+, but mainly by stimulated influx. Blockade of cyclo-oxygenase with aspirin only slightly reduced the [Ca2+]i changes, indicating that thromboxane A2 is not a major mediator of the calcium movements. In control conditions PAF could stimulate shape-change, aggregation and secretion. Aggregation and secretion were roughly halved by blockade of cyclo-oxygenase. Shape-change and secretion still occurred under conditions where the [Ca2+]i rise was small or suppressed, indicating a role for intracellular activators other than Ca2+. The possible involvement of products of phosphoinositide breakdown is discussed.

SUBMITTER: Hallam TJ

PROVIDER: S-EPMC1153410 | biostudies-other | 1984 Mar

REPOSITORIES: biostudies-other

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Project description:Currently available genetically encoded calcium indicators (GECIs) utilize calmodulins (CaMs) or troponin C from metazoa such as mammals, birds, and teleosts, as calcium-binding domains. The amino acid sequences of the metazoan calcium-binding domains are highly conserved, which may limit the range of the GECI key parameters and cause undesired interactions with the intracellular environment in mammalian cells. Here we have used fungi, evolutionary distinct organisms, to derive CaM and its binding partner domains and design new GECI with improved properties. We applied iterative rounds of molecular evolution to develop FGCaMP, a novel green calcium indicator. It includes the circularly permuted version of the enhanced green fluorescent protein (EGFP) sandwiched between the fungal CaM and a fragment of CaM-dependent kinase. FGCaMP is an excitation-ratiometric indicator that has a positive and an inverted fluorescence response to calcium ions when excited at 488 and 405 nm, respectively. Compared with the GCaMP6s indicator in vitro, FGCaMP has a similar brightness at 488 nm excitation, 7-fold higher brightness at 405 nm excitation, and 1.3-fold faster calcium ion dissociation kinetics. Using site-directed mutagenesis, we generated variants of FGCaMP with improved binding affinity to calcium ions and increased the magnitude of FGCaMP fluorescence response to low calcium ion concentrations. Using FGCaMP, we have successfully visualized calcium transients in cultured mammalian cells. In contrast to the limited mobility of GCaMP6s and G-GECO1.2 indicators, FGCaMP exhibits practically 100% molecular mobility at physiological concentrations of calcium ion in mammalian cells, as determined by photobleaching experiments with fluorescence recovery. We have successfully monitored the calcium dynamics during spontaneous activity of neuronal cultures using FGCaMP and utilized whole-cell patch clamp recordings to further characterize its behavior in neurons. Finally, we used FGCaMP in vivo to perform structural and functional imaging of zebrafish using wide-field, confocal, and light-sheet microscopy.

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Stimulus-response coupling in human platelets. Changes evoked by platelet-activating factor in cytoplasmic free calcium monitored with the fluorescent calcium indicator quin2.

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