Calcium uptake and release characteristics of the dense tubules of digitonin-permeabilized human platelets

Wenche Jy, Duncan H. Haynes

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

The kinetics of ATP-driven Ca2+ 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 Ca2+ indicator Quin2 without increasing the passive permeability of the dense tubular membrane for Ca2+. Experimentation was carried out with platelets treated with 3 μg/ml digitonin reisolated and resuspended in detergent-free medium ('digitonin-permeablized' 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°C). The active uptake is inhibited by 15 μM 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+] ≈ Km is increased when the digitonin-permeablized 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 triphosphate (IP3). The maximal extent of release is 50%; the EC50 for IP3 is approx. 12 μM. The data are compared with findings for fractionated dense tubular membrane vesicles and for the intact platelet.

Original languageEnglish
Pages (from-to)374-382
Number of pages9
JournalBBA - Biomembranes
Volume944
Issue number3
DOIs
StatePublished - Oct 20 1988

Fingerprint

Digitonin
Platelets
Blood Platelets
Calcium
Adenosine Triphosphate
Membranes
Chlortetracycline
Trifluoperazine
Inositol
Cell membranes
L-Lactate Dehydrogenase
Detergents
Permeability
Acetates
Blood
Fluorescence
Cell Membrane
Kinetics

Keywords

  • (Human)
  • Calcium ion transport
  • Chlorotetracycline
  • Dense tubule
  • Digitonin
  • Platelet membrane

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Cell Biology

Cite this

Calcium uptake and release characteristics of the dense tubules of digitonin-permeabilized human platelets. / Jy, Wenche; Haynes, Duncan H.

In: BBA - Biomembranes, Vol. 944, No. 3, 20.10.1988, p. 374-382.

Research output: Contribution to journalArticle

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AB - The kinetics of ATP-driven Ca2+ 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 Ca2+ indicator Quin2 without increasing the passive permeability of the dense tubular membrane for Ca2+. Experimentation was carried out with platelets treated with 3 μg/ml digitonin reisolated and resuspended in detergent-free medium ('digitonin-permeablized' 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°C). The active uptake is inhibited by 15 μM 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+] ≈ Km is increased when the digitonin-permeablized 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 triphosphate (IP3). The maximal extent of release is 50%; the EC50 for IP3 is approx. 12 μM. The data are compared with findings for fractionated dense tubular membrane vesicles and for the intact platelet.

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