STIMULATION OF INTRACELLULAR FREE CALCIUM INCREASES BY PLATELET-ACTIVATING-FACTOR IN HT29 COLON-CARCINOMA CELLS - SPECTROFLUOROMETRIC AND PRELIMINARY SPATIOTEMPORAL ANALYSIS USING CONFOCAL LASER-SCANNING FLUORESCENCE IMAGING MICROSCOPY

We examined the ability of platelet-activating factor (PAF) and its ly so derivative (lyso-PAF) to elicit increases in intracellular free cal cium concentration ([Ca2+]i) in HT29 human colon carcinoma cells. Usin g spectrofluorimetric analysis with indo-I as the [Ca2+]i reporter mol ecule, we found that 1-10 muM 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 r eciprocally (heterologous desensitization). The potent PAF receptor an tagonist WEB 2086 [1,41-diazepin-2yl]-1-(4-morpholinyl)-1-propanone} s uccessfully blocked PAF-induced [Ca2+]i elevations, but did not affect rises in response to lyso-PAF, suggesting that lyso-PAF may act throu gh a different cellular receptor or mechanism. Higher concentrations ( >10 muM) of PAF resulted in non-reversible [Ca2+]i elevations which we re caused by Ca2+ influx following membrane lysis. However, the WEB 20 86 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-sensi tive receptors. Preliminary spatio-temporal observations, using confoc al microscopy and fluo-3 as the [Ca2+]i reporter molecule, suggested t hat PAF can also induce [Ca2+]i elevations in the absence of cell lysi s in monolayer HT29 cells. Visual impressions were obtained of cellula r and subcellular heterogeneity and of [Ca2+]i oscillations in respond ing cells. However, these need to be interpreted with caution because of the intrinsic limitations of the methodology, particularly using no n-ratiometric dyes. The significance of a receptor-mediated, reversibl e elevation of [Ca2+]i by sub-toxic concentrations of PAF in HT29 colo n cancer cells remains to be elucidated, but it is tempting to specula te that PAF might function as a locally acting signalling mediator in these and possibly other tumour cells.