Calcium uptake and release characteristics of the dense tubules of digitonin-permeabilized human platelets.
The kinetics of ATP-driven Ca2+ uptake by the dense tubules were studied in digitonin-permeabilized human blood platelets. Digitonin at 3 micrograms/ml was shown capable of permeabilizing the plasma membrane to lactate dehydrogenase and the cytoplasmic Ca2+ indicator Quin2 without increasing the passive permeability of the dense tubular membrane for Ca2+. Experimentation was carried out with platelets treated with 3 micrograms/ml digitonin reisolated and resuspended in detergent-free medium ('digitonin-permeabilized' platelets). Active Ca2+ 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 degrees C). The active uptake is inhibited by 15 microM trifluoperazine. The process is saturable with respect to external [Ca2+], with a Km 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 (Km = 160 +/- 5 nM, n = 1.50 +/- 0.05). The rate of uptake at [Ca2+] approximately Km is increased when the digitonin-permeabilized platelets are preincubated with 100 nM phorbol 12-myristate 13-acetate. Actively accumulated Ca2+ is rapidly released (less than 1 min) by addition of D-myo-inositol trisphosphate (IP3). The maximal extent of release is 50%; the EC50 for IP3 is approx. 12 microM. The data are compared with findings for fractionated dense tubular membrane vesicles and for the intact platelet.
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Department of Pharmacology, University of Miami School of Medicine, FL 33101.
Haynes
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D000255:Adenosine Triphosphate / Q000378:metabolism; D001692:Biological Transport; D001792:Blood Platelets / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002751:Chlortetracycline; D004072:Digitonin / Q000494:pharmacology; D004987:Ethers / Q000494:pharmacology; D015544:Inositol 1,4,5-Trisphosphate; D007295:Inositol Phosphates / Q000494:pharmacology; D007425:Intracellular Membranes / Q000378:metabolism*; D015759:Ionomycin; D007700:Kinetics; D015388:Organelles / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D004987:Ethers; D007295:Inositol Phosphates; D015759:Ionomycin; D015544:Inositol 1,4,5-Trisphosphate; D000255:Adenosine Triphosphate; D004072:Digitonin; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; D002751:Chlortetracycline