A fluorescent hydrophobic probe used for monitoring the kinetics of exocytosis phenomena

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Abstract
A fluorescence method is presented for quantitatively analyzing exocytosis phenomena and monitoring their kinetics. The method is based on the particular properties of a hydrophobic fluorescent probe, 1-[4-(trimethylammonio)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH) [Prendergast, F. G., Haugland, R. P., and Callahan, P. J. (1981) Biochemistry 20, 7333-7338; Kuhry, J. G., Fonteneau, P., Duportail, G., Maechling, C., and Laustriat, G. (1983) Cell Biophys, 5, 129-140; Kuhry, J. G., Duportail, G., Bronner, C., and Laustriat, G. (1985) Biochim. Biophys. Acta 845, 60-67]. When this probe is interacted with intact resting cells in aqueous suspensions, it labels solely the membranes that are in contact with the external medium and is incorporated into them according to a partition equilibrium; i.e., the amount of the probe incorporated is proportional to the available membrane surface. TMA-DPH is highly fluorescent in membranes and not at all in water. Thus, a measurement of the TMA-DPH fluorescence intensity provides a signal proportional to the membrane surface. In secretory cells, the membrane surface available for the probe is increased upon fusion of the membrane of the secretory granules with the cell plasma membranes, directly or via intergranule fusion. Thus, when these cells are stimulated, more TMA-DPH is incorporated than in resting cells since the probe is allowed to also interact with the granule membranes now connected with the external medium by pores. This process results in a proportional increase in the TMA-DPH fluorescence intensity. The response was found to be very rapid and able to follow accurately the exocytosis kinetics. The method was tested on the histamine release process induced from peritoneal rat mast cells by compound 48/80 and ionomycin and from 2H3 rat basophilic leukemia cells (RBL) by ionomycin. The TMA-DPH fluorescence signal was compared with the histamine level, and the validity of the method was controlled by kinetic analyzes, dose-response studies, and response to metabolic inhibitors and to temperature changes. The conclusions were corroborated by a comparative study with the permeant probe 1,6-diphenylhexa-1,3,5-triene (DPH), and its principle was supported by fluorescence microphotography results. Under Discussion, arguments are developed to establish the specificity of the method for exocytosis and to discard the contribution of other processes such as the increase in the membrane permeability, labeling or leaking of secreted products, or changes in membrane potential. Finally, the method appeared to be of value to study thoroughly secretory systems, allowing differentiation of exocytosis from other secretory mechanisms.
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