Biology Reference
In-Depth Information
A
B
1.0
1.0
35C
0.9
30 C
0.9
0.8
0.8
Proben.
0.7
35 C
Sulfin.
0.7
0.6
No
inhibitor
0.5
0.6
0
10
20
30
0
5
10 15
Time (min)
20
25
Time (min)
Fig. 6 (A) Time course of indicator loss at 30 and 35 C from REF52 cells loaded with Fura-2. (B)
E
ect of probenecid (1 mM) and sulfinpyrazone (75 m M) on indicator extrusion from REF52 cells at
35 C; arrow marks the time of drug application. For all measurements, the cells were loaded with Fura-
2 by incubation with AM ester/Pluronic dispersion in HEPES-bu V ered HBSS at 25 C. Experiments
were done in HBSS at the specified temperatures. Total Fura-2 fluorescence (F T ¼ F 340 þ F 380 )is
monitored over time. Each trace was normalized by dividing by the value of F T at time 0.
V
C. Dye Leakage or Extrusion from Cells
Once an indicator is loaded into cells, it leaks out at a rate that is strongly tempera-
ture-dependent. For mammalian cells, the loss rate is maximal at 37 Canddropso
V
sharply as temperature is lowered, 11 as shown in Fig. 6 A. Loss of indicator occurs by
an extrusion mechanism for organic anions ( Di Virgilio et al.,1988 ) and can be
blocked e
ectively by inhibitors of uric acid transport such as probenecid and
sulfinpyrazone ( Di Virgilio et al., 1988, 1990 ), as illustrated in Fig. 6 B. Sulfinpyrazone
at tens to hundreds of micromolar has been found to be e
V
ective, whereas probenecid
works at millimolar dosage. High concentrations of inhibitor virtually can stop
indicator extrusion but also can induce signs of cellular stress such as blebbing.
Therefore, using the minimum concentration of inhibitor necessary to reduce the
rate of indicator loss to a tolerable level without attempting to block the process
altogether is advisable. In general, slowing indicator loss by lowering the temperature
at which experiments are conducted is preferable to using transport inhibitors.
V
11
The sharp increase in rate of indicator leakage with rising temperature is another reason why
compartmentalization is a more severe problem when cells are incubated with AM esters at higher
temperatures. Higher temperature accelerates loss of dye from the cytosol but not from organelles, so
compartmentalized dye becomes a larger proportion of the total intracellular dye content.
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