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. 1989 Mar 15;258(3):923–926. doi: 10.1042/bj2580923

Receptor-mediated calcium entry in fura-2-loaded human platelets stimulated with ADP and thrombin. Dual-wavelengths studies with Mn2+.

S O Sage 1, J E Merritt 1, T J Hallam 1, T J Rink 1
PMCID: PMC1138454  PMID: 2730577

Abstract

Previous studies of the early kinetics of rises in cytosolic free [Ca2+] in fura-2-loaded human platelets suggested that: (1) Ca2+ entry slightly preceded internal discharge with thrombin and other agonists known to promote inositol lipid hydrolysis; (2) with ADP, Ca2+ entry occurred without measurable delay and clearly preceded internal Ca2+ discharge. In the present work, Mn2+ added to the external medium was used as a marker for Ca2+ entry. By using an excitation wavelength of 360 nm, a quench of fura-2 can be followed to report Mn2+ entry without 'contamination' of the signal by changes in [Ca2+], because at this isosbestic wavelength Ca2+ does not alter fura-2 fluorescence. The present results show that, with thrombin stimulation, readily discernible Mn2+ entry starts after discharge of internal Ca2+ and is maintained for many minutes. With ADP, Mn2+ entry starts without measurable delay (less than 20 ms) and clearly precedes internal Ca2+ discharge. However, the enhanced Mn2+ permeability is only short-lived. These results, considered alongside previous data, point to the possible presence of at least three different receptor-mediated Ca2+-entry mechanisms in human platelets, one of which may include regulation by the 'state of filling' of this dischargeable Ca2+ store.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Berridge M. J. The Croonian lecture, 1988. Inositol lipids and calcium signalling. Proc R Soc Lond B Biol Sci. 1988 Sep 22;234(1277):359–378. doi: 10.1098/rspb.1988.0054. [DOI] [PubMed] [Google Scholar]
  2. Grynkiewicz G., Poenie M., Tsien R. Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985 Mar 25;260(6):3440–3450. [PubMed] [Google Scholar]
  3. Hallam T. J., Jacob R., Merritt J. E. Evidence that agonists stimulate bivalent-cation influx into human endothelial cells. Biochem J. 1988 Oct 1;255(1):179–184. doi: 10.1042/bj2550179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Hallam T. J., Rink T. J. Agonists stimulate divalent cation channels in the plasma membrane of human platelets. FEBS Lett. 1985 Jul 8;186(2):175–179. doi: 10.1016/0014-5793(85)80703-1. [DOI] [PubMed] [Google Scholar]
  5. Hallam T. J., Rink T. J. Responses to adenosine diphosphate in human platelets loaded with the fluorescent calcium indicator quin2. J Physiol. 1985 Nov;368:131–146. doi: 10.1113/jphysiol.1985.sp015850. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Hallam T. J., Sanchez A., Rink T. J. Stimulus-response coupling in human platelets. Changes evoked by platelet-activating factor in cytoplasmic free calcium monitored with the fluorescent calcium indicator quin2. Biochem J. 1984 Mar 15;218(3):819–827. doi: 10.1042/bj2180819. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hallam T. J., Thompson N. T., Scrutton M. C., Rink T. J. The role of cytoplasmic free calcium in the responses of quin2-loaded human platelets to vasopressin. Biochem J. 1984 Aug 1;221(3):897–901. doi: 10.1042/bj2210897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Merritt J. E., Hallam T. J. Platelets and parotid acinar cells have different mechanisms for agonist-stimulated divalent cation entry. J Biol Chem. 1988 May 5;263(13):6161–6164. [PubMed] [Google Scholar]
  9. Merritt J. E., Rink T. J. Regulation of cytosolic free calcium in fura-2-loaded rat parotid acinar cells. J Biol Chem. 1987 Dec 25;262(36):17362–17369. [PubMed] [Google Scholar]
  10. Putney J. W., Jr A model for receptor-regulated calcium entry. Cell Calcium. 1986 Feb;7(1):1–12. doi: 10.1016/0143-4160(86)90026-6. [DOI] [PubMed] [Google Scholar]
  11. Rink T. J. A real receptor-operated calcium channel? Nature. 1988 Aug 25;334(6184):649–650. doi: 10.1038/334649a0. [DOI] [PubMed] [Google Scholar]
  12. Rink T. J., Smith S. W., Tsien R. Y. Cytoplasmic free Ca2+ in human platelets: Ca2+ thresholds and Ca-independent activation for shape-change and secretion. FEBS Lett. 1982 Nov 1;148(1):21–26. doi: 10.1016/0014-5793(82)81234-9. [DOI] [PubMed] [Google Scholar]
  13. Sage S. O., Rink T. J. Kinetic differences between thrombin-induced and ADP-induced calcium influx and release from internal stores in fura-2-loaded human platelets. Biochem Biophys Res Commun. 1986 May 14;136(3):1124–1129. doi: 10.1016/0006-291x(86)90450-x. [DOI] [PubMed] [Google Scholar]
  14. Sage S. O., Rink T. J. The kinetics of changes in intracellular calcium concentration in fura-2-loaded human platelets. J Biol Chem. 1987 Dec 5;262(34):16364–16369. [PubMed] [Google Scholar]
  15. Zschauer A., van Breemen C., Bühler F. R., Nelson M. T. Calcium channels in thrombin-activated human platelet membrane. Nature. 1988 Aug 25;334(6184):703–705. doi: 10.1038/334703a0. [DOI] [PubMed] [Google Scholar]

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