title: 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.
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.
authors.affiliation: MRC Unit of Clinical Oncology and Radiotherapeutics, MRC Centre, Cambridge, U.K.
authors.forename: M
authors.initials: M
authors.lastname: Lohmeyer
authors.forename: L
authors.initials: L
authors.lastname: McNaughton
authors.forename: S P
authors.initials: SP
authors.lastname: Hunt
authors.forename: P
authors.initials: P
authors.lastname: Workman
mesh_terms: D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D003110:Colonic Neoplasms / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008856:Microscopy, Fluorescence / Q000379:methods; D010972:Platelet Activating Factor / Q000494:pharmacology / Q000502:physiology*; D013050:Spectrometry, Fluorescence; D014407:Tumor Cells, Cultured
chemical_list: D010972:Platelet Activating Factor; D002118:Calcium