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Biochem Pharmacol. 1994 Mar 15;47(6):975-85.

Stimulation of intracellular free calcium increases by platelet-activating factor in HT29 colon carcinoma cells. Spectrofluorimetric and preliminary spatio-temporal analysis using confocal laser scanning fluorescence imaging microscopy.

Author information

1
MRC Unit of Clinical Oncology and Radiotherapeutics, MRC Centre, Cambridge, U.K.

Abstract

We examined the ability of platelet-activating factor (PAF) and its lyso derivative (lyso-PAF) to elicit increases in intracellular free calcium concentration ([Ca2+]i) in HT29 human colon carcinoma cells. Using spectrofluorimetric analysis with indo-1 as the [Ca2+]i reporter molecule, we found that 1-10 microM concentrations of both lipids stimulated substantial, reversible, monophasic [Ca2+]i elevations. Evidence was obtained that the two lipids may act via specific receptors to release Ca2+ from internal stores. Homologous desensitization was observed in both cases and PAF and lyso-PAF were also able to desensitize cells reciprocally (heterologous desensitization). The potent PAF receptor antagonist WEB 2086 (3-[4-(chlorophenyl)-9-methyl-6H-thieno[3,2-f][1,2, 4]triazolo-[4,3-a][1,4]-diazepin-2-yl]-1-(4-morpholinyl)-1-propano ne) successfully blocked PAF-induced [Ca2+]i elevations, but did not affect rises in response to lyso-PAF, suggesting that lyso-PAF may act through a different cellular receptor or mechanism. Higher concentrations (> 10 microM) of PAF resulted in non-reversible [Ca2+]i elevations which were caused by Ca2+ influx following membrane lysis. However, the WEB 2086 insensitivity of these effects and the resultant cellular toxicity clearly showed that such events were mechanistically distinct from the reversible [Ca2+]i elevations apparently operating via WEB 2086-sensitive receptors. Preliminary spatio-temporal observations, using confocal microscopy and fluo-3 as the [Ca2+]i reporter molecule, suggested that PAF can also induce [Ca2+]i elevations in the absence of cell lysis in monolayer HT29 cells. Visual impressions were obtained of cellular and subcellular heterogeneity and of [Ca2+]i oscillations in responding cells. However, these need to be interpreted with caution because of the intrinsic limitations of the methodology, particularly using non-ratiometric dyes. The significance of a receptor-mediated, reversible elevation of [Ca2+]i by sub-toxic concentrations of PAF in HT29 colon cancer cells remains to be elucidated, but it is tempting to speculate that PAF might function as a locally acting signalling mediator in these and possibly other tumour cells.

PMID:
8147917
DOI:
10.1016/0006-2952(94)90408-1
[Indexed for MEDLINE]

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