Abstract
The kinetics of ATP-driven Ca
2+ uptake by the dense tubules were studied in digitonin-permeablized human blood platelets. Digitonin at 3 μg/ml was shown capable of permeablizing the plasma membrane to lactate dehydrogenase and the cytoplasmic Ca
2+ indicator Quin2 without increasing the passive permeability of the dense tubular membrane for Ca
2+. Experimentation was carried out with platelets treated with 3 μg/ml digitonin reisolated and resuspended in detergent-free medium (‘digitonin-permeablized’ platelets). Active Ca
2+ accumulation, which occurs over a period of minutes, was monitored by the increase in the fluorescence of chlorotetracycline after the addition of Mg-ATP (37°C). The active uptake is inhibited by 15 μM trifluoperazine. The process is saturable with respect to external [Ca
2+], with a
K
m of 180 ± 5 nM and a Hill coefficient (n) of 1.40 ± 0.05. Analysis of the maximal uptake in steady state gave similar results (
K
m = 160 ± 5 nM,
n = 1.50 ± 0.05). The rate of uptake at [Ca
2+] ≈
K
m is increased when the digitonin-permeablized platelets are preincubated with 100 nM phorbol 12-myristate 13-acetate. Actively accumulated Ca
2+ is rapidly released (less than 1 min) by addition of
d
-myo-
inositol
triphosphate (IP
3). The maximal extent of release is 50%; the EC
50 for IP
3 is approx. 12 μM. The data are compared with findings for fractionated dense tubular membrane vesicles and for the intact platelet.
