The effect of atp-induced permeabilization on loading of the na+
probe, sbfi, into endothelial cells.
Cutaia, Michael, Ryan Davis, Nancy Parks, Sharon Rounds.
Providence VA Medical Center, Pulmonary Disease Division,
Department of Medicine, Brown University School of Medicine,
Providence, R.I., 02908
APStracts 3:0122A, 1996.
The fluoroprobe, sodium-binding benzofuran isophthalate (SBFI), is
used to measure intracellular cytosolic sodium concentration [Nai]. A
problem with the use of this probe is the difficulty in loading it
into cells. ATP reversibly increases membrane permeability of some
cells via activation of ATP4- receptors. We investigated the effect
of ATP-induced membrane permeabilization on loading of the
acetoxymethyl ester form of SBFI (SBFI/AM) into bovine pulmonary
artery endothelial cells (BPAEC). Monolayers were incubated in a
series of solutions which reversibly opened pores, loaded the
fluoroprobe, and finally sealed the pores. ATP (1-5mM) or 3'-O-(4
-Benzoyl)benzoyl- adenosine 5'-triphosphate (BzATP; 0.1-1mM), an
analogue 30-100x more specific for ATP4- receptors, were utilized to
permeabilize the cell membrane. The signal/background ratio (S/B) of
the intracellular SBFI fluorescent signal was used as an indicator of
the effectiveness of dye loading. ATP and BzATP significantly
increased the S/B ratio compared to the values obtained using the
standard dye-loading procedure without ATP, indicating that
permeabilization increased SBFI/AM entry into cells. The
permeabilization procedure produced a small decrease in cell
viability, as determined with a fluorescent viability assay (ethidium
dimer uptake), compared to the standard method of loading SBFI/AM. We
used the procedure to measure baseline [Nai] and changes in [Nai]
following the administration of ouabain (10-4M) and monensin (10-5M).
Baseline [Nai] with this procedure (19.7+/- 2.7mM; n=15 monolayers)
was similar to measurements made in other cell types with the
standard method of loading the probe. We conclude: 1) the ATP-induced
permeabilization technique is an improved dye-loading method for
SBFI/AM in endothelial cell monolayers which facilitates measurement
of [Nai]; and 2) these data suggest the presence of an ATP4- pore
-forming mechanism in this cell type.
Received 1 June 1995; accepted in final form 14 February 1996.
APS Manuscript Number A571-5.
Article publication pending Journal of Applied Physiology.
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 13 March 96