Skip to main content
NCBI home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1986 Mar;77(3):878–886. doi: 10.1172/JCI112385

Effect of common agonists on cytoplasmic ionized calcium concentration in platelets. Measurement with 2-methyl-6-methoxy 8-nitroquinoline (quin2) and aequorin.

J A Ware, P C Johnson, M Smith, E W Salzman
PMCID: PMC423474  PMID: 3081576

Abstract

Because of controversy regarding the relationship of cytoplasmic ionized calcium concentration ([Cai2+]) to platelet activation, we studied the correlation of platelet aggregation and ATP secretion with [Cai2+] as determined by 2-methyl-6-methoxy 8-nitroquinoline (quin2) and aequorin in response to ADP, epinephrine, collagen, the Ca2+ ionophore A23187, and thrombin. Both indicators showed a concentration-dependent increase in [Cai2+] in response to all agonists except epinephrine when gel-filtered platelets were suspended in media containing 1 mM Ca2+. With epinephrine, a rise in [Cai2+] was indicated by aequorin, but not by quin2; [Cai2+] signals, aggregation, and secretion were suppressed by EGTA. ADP [0.5 microM] produced a rise in [Cai2+] that was registered by both aequorin and quin2 in platelets in Ca2+-containing media; addition of EGTA to the medium raised the threshold concentration of ADP to 5.0 microM for both indicators. Collagen produced progressive concentration-related increases in [Cai2+] and aggregation in aspirin-treated aequorin-loaded platelets. Quin2 failed to indicate a rise in [Cai2+]at lower collagen concentrations with EGTA or aspirin. [Cai2+] response to A23187 and thrombin was reduced by addition of EGTA to platelets loaded with either aequorin or quin2. With all five agonists in all conditions tested, aequorin [Cai2+] signals occurred at the same agonist concentration as that or lower than that which produced platelet shape change, aggregation, or secretion. Platelet activation was better correlated with changes in [Cai2+] indicated by aequorin than with the response of quin2, possibly because aequorin is more sensitive to local zones of [Cai2+] elevation.

Full text

PDF
878

Selected References

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

  1. Akkerman J. W., Niewiarowski S., Holmsen H. Identification of granular heterogeneity in blood platelets by controlled digitonin-induced cell lysis. Thromb Res. 1980 Jan 1;17(1-2):249–254. doi: 10.1016/0049-3848(80)90311-4. [DOI] [PubMed] [Google Scholar]
  2. Ardlie N. G. Calcium ions, drug action and platelet function. Pharmacol Ther. 1982;18(2):249–270. doi: 10.1016/0163-7258(82)90069-9. [DOI] [PubMed] [Google Scholar]
  3. Benton A. M., Gerrard J. M., Michiel T., Kindom S. E. Are lysophosphatidic acids or phosphatidic acids involved in stimulus activation coupling in platelets? Blood. 1982 Sep;60(3):642–649. [PubMed] [Google Scholar]
  4. Blinks J. R., Wier W. G., Hess P., Prendergast F. G. Measurement of Ca2+ concentrations in living cells. Prog Biophys Mol Biol. 1982;40(1-2):1–114. doi: 10.1016/0079-6107(82)90011-6. [DOI] [PubMed] [Google Scholar]
  5. Brass L. F., Shattil S. J. Identification and function of the high affinity binding sites for Ca2+ on the surface of platelets. J Clin Invest. 1984 Mar;73(3):626–632. doi: 10.1172/JCI111252. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Broekman M. J. Phosphatidylinositol 4,5-bisphosphate may represent the site of release of plasma membrane-bound calcium upon stimulation of human platelets. Biochem Biophys Res Commun. 1984 Apr 16;120(1):226–231. doi: 10.1016/0006-291x(84)91437-2. [DOI] [PubMed] [Google Scholar]
  7. DeFeo T. T., Morgan K. G. Calcium-force relationships as detected with aequorin in two different vascular smooth muscles of the ferret. J Physiol. 1985 Dec;369:269–282. doi: 10.1113/jphysiol.1985.sp015900. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Feinman R. D., Detwiler T. C. Platelet secretion induced by divalent cation ionophores. Nature. 1974 May 10;249(453):172–173. doi: 10.1038/249172a0. [DOI] [PubMed] [Google Scholar]
  9. Feinman R. D., Lubowsky J., Charo I., Zabinski M. P. The lumi-aggregometer: a new instrument for simultaneous measurement of secretion and aggregation by platelets. J Lab Clin Med. 1977 Jul;90(1):125–129. [PubMed] [Google Scholar]
  10. Feinstein M. B., Fraser C. Human platelet secretion and aggregation induced by calcium ionophores. Inhibition by PGE1 and dibutyryl cyclic AMP. J Gen Physiol. 1975 Nov;66(5):561–581. doi: 10.1085/jgp.66.5.561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Flatman P. W. The effect of buffer composition and deoxygenation on the concentration of ionized magnesium inside human red blood cells. J Physiol. 1980 Mar;300:19–30. doi: 10.1113/jphysiol.1980.sp013148. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Harary H. H., Brown J. E. Spatially nonuniform changes in intracellular calcium ion concentrations. Science. 1984 Apr 20;224(4646):292–294. doi: 10.1126/science.6710144. [DOI] [PubMed] [Google Scholar]
  13. Hawiger J., Parkinson S., Timmons S. Prostacyclin inhibits mobilisation of fibrinogen-binding sites on human ADP- and thrombin-treated platelets. Nature. 1980 Jan 10;283(5743):195–197. doi: 10.1038/283195a0. [DOI] [PubMed] [Google Scholar]
  14. Holmsen H., Dangelmaier C. A. Evidence that the platelet plasma membrane is impermeable to calcium and magnesium complexes of A23187. A23187-induced secretion is inhibited by MG2+ and Ca2+, and requires aggregation and active cyclooxygenase. J Biol Chem. 1981 Oct 25;256(20):10449–10452. [PubMed] [Google Scholar]
  15. Johnson P. C., Ware J. A., Cliveden P. B., Smith M., Dvorak A. M., Salzman E. W. Measurement of ionized calcium in blood platelets with the photoprotein aequorin. Comparison with Quin 2. J Biol Chem. 1985 Feb 25;260(4):2069–2076. [PubMed] [Google Scholar]
  16. Kajikawa N., Kaibuchi K., Matsubara T., Kikkawa U., Takai Y., Nishizuka Y., Itoh K., Tomioka C. A possible role of protein kinase C in signal-induced lysosomal enzyme release. Biochem Biophys Res Commun. 1983 Oct 31;116(2):743–750. doi: 10.1016/0006-291x(83)90587-9. [DOI] [PubMed] [Google Scholar]
  17. Knight D. E., Hallam T. J., Scrutton M. C. Agonist selectivity and second messenger concentration in Ca2+-mediated secretion. Nature. 1982 Mar 18;296(5854):256–257. doi: 10.1038/296256a0. [DOI] [PubMed] [Google Scholar]
  18. Knight D. E., Scrutton M. C. Direct evidence for a role for Ca2+ in amine storage granule secretion by human platelets. Thromb Res. 1980 Nov 15;20(4):437–446. doi: 10.1016/0049-3848(80)90282-0. [DOI] [PubMed] [Google Scholar]
  19. Lapetina E. G. Metabolism of inositides and the activation of platelets. Life Sci. 1983 May 2;32(18):2069–2082. doi: 10.1016/0024-3205(83)90094-2. [DOI] [PubMed] [Google Scholar]
  20. Lapetina E. G., Siess W. The role of phospholipase C in platelet responses. Life Sci. 1983 Sep 12;33(11):1011–1018. doi: 10.1016/0024-3205(83)90654-9. [DOI] [PubMed] [Google Scholar]
  21. Lawson D., Fewtrell C., Raff M. C. Localized mast cell degranulation induced by concanavalin A-sepharose beads. Implications for the Ca2+ hypothesis of stimulus-secretion coupling. J Cell Biol. 1978 Nov;79(2 Pt 1):394–400. doi: 10.1083/jcb.79.2.394. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Le Breton G. C., Sandler W. C., Feinberg H. The effect of D2O and chlortetracycline on ADP-induced platelet shape change and aggregation. Thromb Res. 1976 Apr;8(4):477–485. doi: 10.1016/0049-3848(76)90225-5. [DOI] [PubMed] [Google Scholar]
  23. Lloyd J. V., Nishizawa E. E., Haldar J., Mustard J. F. Changes in 32 p-labelling of platelet phospholipids in response to ADP. Br J Haematol. 1972 Nov;23(5):571–585. doi: 10.1111/j.1365-2141.1972.tb07092.x. [DOI] [PubMed] [Google Scholar]
  24. Massini P., Lüscher E. F. On the significance of the influx of calcium ions into stimulated human blood platelets. Biochim Biophys Acta. 1976 Jul 1;436(3):652–663. doi: 10.1016/0005-2736(76)90447-8. [DOI] [PubMed] [Google Scholar]
  25. Nishizuka Y. The role of protein kinase C in cell surface signal transduction and tumour promotion. Nature. 1984 Apr 19;308(5961):693–698. doi: 10.1038/308693a0. [DOI] [PubMed] [Google Scholar]
  26. Owen N. E., Feinberg H., Le Breton G. C. Epinephrine induces Ca2+ uptake in human blood platelets. Am J Physiol. 1980 Oct;239(4):H483–H488. doi: 10.1152/ajpheart.1980.239.4.H483. [DOI] [PubMed] [Google Scholar]
  27. Rink T. J., Sanchez A., Hallam T. J. Diacylglycerol and phorbol ester stimulate secretion without raising cytoplasmic free calcium in human platelets. Nature. 1983 Sep 22;305(5932):317–319. doi: 10.1038/305317a0. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Rink T. J., Tsien R. Y., Pozzan T. Cytoplasmic pH and free Mg2+ in lymphocytes. J Cell Biol. 1982 Oct;95(1):189–196. doi: 10.1083/jcb.95.1.189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rittenhouse-Simmons S., Deykin D. The mobilization of arachidonic acid in platelets exposed to thrombin or ionophore A23187. Effects of adenosine triphosphate deprivation. J Clin Invest. 1977 Aug;60(2):495–498. doi: 10.1172/JCI108801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Rittenhouse-Simmons S. Differential activation of platelet phospholipases by thrombin and ionophore A23187. J Biol Chem. 1981 May 10;256(9):4153–4155. [PubMed] [Google Scholar]
  32. Rose B., Loewenstein W. R. Calcium ion distribution in cytoplasm visualised by aequorin: diffusion in cytosol restricted by energized sequestering. Science. 1975 Dec 19;190(4220):1204–1206. doi: 10.1126/science.1198106. [DOI] [PubMed] [Google Scholar]
  33. Scrutton M. C., Egan C. M. Divalent cation requirements for aggregation of human blood platelets and the role of the anti-coagulant. Thromb Res. 1979;14(4-5):713–727. doi: 10.1016/0049-3848(79)90127-0. [DOI] [PubMed] [Google Scholar]
  34. Sheu S. S., Sharma V. K., Banerjee S. P. Measurement of cytosolic free calcium concentration in isolated rat ventricular myocytes with quin 2. Circ Res. 1984 Dec;55(6):830–834. doi: 10.1161/01.res.55.6.830. [DOI] [PubMed] [Google Scholar]
  35. Snowdowne K. W., Borle A. B. Measurement of cytosolic free calcium in mammalian cells with aequorin. Am J Physiol. 1984 Nov;247(5 Pt 1):C396–C408. doi: 10.1152/ajpcell.1984.247.5.C396. [DOI] [PubMed] [Google Scholar]
  36. Snowdowne K. W., Ertel R. J., Borle A. B. Measurement of cytosolic calcium with aequorin in dispersed rat ventricular cells. J Mol Cell Cardiol. 1985 Mar;17(3):233–241. doi: 10.1016/s0022-2828(85)80006-7. [DOI] [PubMed] [Google Scholar]
  37. Streb H., Irvine R. F., Berridge M. J., Schulz I. Release of Ca2+ from a nonmitochondrial intracellular store in pancreatic acinar cells by inositol-1,4,5-trisphosphate. Nature. 1983 Nov 3;306(5938):67–69. doi: 10.1038/306067a0. [DOI] [PubMed] [Google Scholar]
  38. Tsien R. Y. New calcium indicators and buffers with high selectivity against magnesium and protons: design, synthesis, and properties of prototype structures. Biochemistry. 1980 May 27;19(11):2396–2404. doi: 10.1021/bi00552a018. [DOI] [PubMed] [Google Scholar]
  39. Tsien R. Y., Pozzan T., Rink T. J. Calcium homeostasis in intact lymphocytes: cytoplasmic free calcium monitored with a new, intracellularly trapped fluorescent indicator. J Cell Biol. 1982 Aug;94(2):325–334. doi: 10.1083/jcb.94.2.325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Tsien R. Y., Rink T. J., Poenie M. Measurement of cytosolic free Ca2+ in individual small cells using fluorescence microscopy with dual excitation wavelengths. Cell Calcium. 1985 Apr;6(1-2):145–157. doi: 10.1016/0143-4160(85)90041-7. [DOI] [PubMed] [Google Scholar]
  41. Young J. D., Ko S. S., Cohn Z. A. The increase in intracellular free calcium associated with IgG gamma 2b/gamma 1 Fc receptor-ligand interactions: role in phagocytosis. Proc Natl Acad Sci U S A. 1984 Sep;81(17):5430–5434. doi: 10.1073/pnas.81.17.5430. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Zavoico G. B., Feinstein M. B. Cytoplasmic Ca2+ in platelets is controlled by cyclic AMP: antagonism between stimulators and inhibitors of adenylate cyclase. Biochem Biophys Res Commun. 1984 Apr 30;120(2):579–585. doi: 10.1016/0006-291x(84)91294-4. [DOI] [PubMed] [Google Scholar]
  43. Zucker M. B., Grant R. A. Nonreversible loss of platelet aggregability induced by calcium deprivation. Blood. 1978 Sep;52(3):505–513. [PubMed] [Google Scholar]

Articles from Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation

RESOURCES