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., & Callahan, P.J. (1981) Biochemistry 20, 7333-7338; Kuhry, J.G., Fonteneau, P., Duportail, G., Maechling, C., & Laustriat, G. (1983) Cell Biophys. 5, 129-140; Kuhry, J.G., Duportail, G., Bronner, C., & 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.(ABSTRACT TRUNCATED AT 250 WORDS)
journal_name
Biochemistryjournal_title
Biochemistryauthors
Bronner C,Landry Y,Fonteneau P,Kuhry JGdoi
10.1021/bi00356a045subject
Has Abstractpub_date
1986-04-22 00:00:00pages
2149-54issue
8eissn
0006-2960issn
1520-4995journal_volume
25pub_type
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