Cardiovascular Drugs and Therapy

, Volume 9, Issue 6, pp 815–821 | Cite as

Blockade of receptor-operated calcium channels by mibefradil (Ro 40-5967): Effects on intracellular calcium and platelet aggregation

  • Martina Hahn
  • Vsevolod A. Tkachuk
  • Valery N. Bochkov
  • Ivan B. Cheglakov
  • Jean-Paul Clozel
Experimental Pharmacology

Summary

The goal of the present study was to evaluate if mibefradil, a novel nondihydropyridine Ca2+ antagonist, could block receptor-operated calcium channels (ROCC) present in human platelets and to determine the functional consequences of this blockade. Therefore, the effect of mibefradil on increases in intracellular Ca2+ concentrations and aggregation of human platelets induced by platelet activating factor (PAF) was examined. In order to differentiate effects on Ca2+ mobilization from intracellular stores from those on Ca2+ influx through ROCC, intracellular Ca2+ concentrations were measured either in fura-2-loaded platelets or in cells loaded with both BAPTA and fura-2. Mibefradil totally and dose dependently inhibited PAF-induced Ca2+ influx with a maximal effective concentration of 10 µM, but at this concentration only reduced Ca2+ mobilization from intracellular stores. A similar effect was observed when platelets were stimulated with ADP, suggesting that mibefradil was indeed interfering with ROCC and not specifically with PAF receptors. In the same range of concentrations, mibefradil inhibited Ca2+-dependent platelet aggregation induced by PAF. This effect was most likely due to the inhibition of ROCC, as Ca2+-independent aggregation induced by phorbolmyristyl-acetate (PMA) was insensitive to mibefradil. We conclude that mibefradil, which has previously been described to be an antagonist for L- and T-Type Ca2+ channels, also blocks receptor-operated Ca2+ channels. This blockade seems to be functionally relevant for platelet aggregation.

Key Words

mibefradil human platelets fura-2 BAPTA receptor operated calcium channels voltage operated calcium channels 

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References

  1. 1.
    Mishra SK, Hermsmeyer K. Selective inhibition of T-type Ca2+ channels by Ro 40-5967.Circ Res 1994;75:144–148.PubMedGoogle Scholar
  2. 2.
    Lacinova L, Welling A, Bosse E, Ruth P, Flockerzi V, Hofmann F. Interaction of Ro 40-5967 with the stable expressed alphal subunit of the cardiac L-type calcium channel. Submitted.Google Scholar
  3. 3.
    Clozel J-P, Osterrieder W, Kleinbloesem CH, et al. Ro 40-5967: A new nondihydropyridine calcium antagonist.Cardiovasc Drug Rev 1991;9:4–17.Google Scholar
  4. 4.
    Avdonin PV, Men'shikov M, Svitina UI, Tkachuk VA. Blocking of the receptor-stimulated calcium entry into human platelets by verapamil and nicardipine.Thromb Res 1988;52:587–597.PubMedGoogle Scholar
  5. 5.
    Blache D, Ciavatti M, Ojeda C. The effect of calcium channel blockers on blood platelet function, especially calcium uptake.Biochim Biophys Acta 1987;923:401–412.PubMedGoogle Scholar
  6. 6.
    Lee TC, Malone B, Snyder F. Stimulation of calcium uptake by 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (plateletactivating factor) in rabbit platelets: Possible involvement of the lipoxygenase pathway.Arch Biochem Biophys 1983;223:33–39.PubMedGoogle Scholar
  7. 7.
    Blache D, Ciavatti M, Ojeda C. Platelet aggregation and endogenous 5-HT secretion in presence of Ca2+, Sr2+ and Ba2+. Effects of calcium antagonists.Thromb Res 1987;46:779–791.PubMedGoogle Scholar
  8. 8.
    Johnsson H. Effects of nifedipine (Adalat®) on platelet function in vitro and in vivo.Thromb Res 1981;21:523–528.PubMedGoogle Scholar
  9. 9.
    MacIntyre DE, Shaw AM. Phospholipid-induced human platelet activation: Effects of calcium channel blockers and calcium chelators.Thromb Res 1983;31:833–844.PubMedGoogle Scholar
  10. 10.
    Mehta J, Mehta P, Ostrowski N, Crews F. Effects of verapamil on platelet aggregation, ATP release and thromboxane generation.Thromb Res 1983;30:469–475.PubMedGoogle Scholar
  11. 11.
    Valone FH. Inhibition of platelet-activating factor binding to human platelets by calcium channel blockers.Thromb Res 1987;45:427–435.PubMedGoogle Scholar
  12. 12.
    Vinge E, Andersson TL, Larsson B. Effects of some calcium antagonists on aggregation by adrenalin and serotonin and on alpha-adrenoceptor radioligand binding in human platelets.Acta Physiol Scand 1988;133:407–416.PubMedGoogle Scholar
  13. 13.
    Wade PJ, Lunt DO, Lad N, Tuffin DP, McCullagh KG. Effect of calcium and calcium antagonists on [3H]-PAF-acether binding to washed human platelets.Thromb Res 1986;41:251–262.PubMedGoogle Scholar
  14. 14.
    Pannocchia A, Praloran N, Arduino C, et al. Absence of (−)[3H]desmethoxyverapamil binding sites on human platelets and lack of evidence for voltage-dependent calcium channels.Eur J Pharmacol 1987;142:83–91.PubMedGoogle Scholar
  15. 15.
    Avdonin PV, Cheglakov IB, Boogry EM, Svitina UI, Mazaev AV, Tkachuk VA. Evidence for the receptor-operated calcium channels in human platelet plasma membrane.Thromb Res 1987;46:29–37.PubMedGoogle Scholar
  16. 16.
    Tsien RY, Pozzan T, Rink TJ. Calcium homeostasis in intact lymphocytes: Cytoplasmic free calcium monitored with a new, intracellularly trapped fluorescent indicator.J Cell Biol 1982;94:325–334.PubMedGoogle Scholar
  17. 17.
    Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties.J Biol Chem 1985;260:3440–3450.PubMedGoogle Scholar
  18. 18.
    Gabbasov ZA, Popov EG, Gavrilov I, Pozin E. Platelet aggregation: The use of optical density fluctuations to study microaggregate formation in platelet suspension.Thromb Res 1989;54:215–223.PubMedGoogle Scholar
  19. 19.
    Avdonin PV, Cheglakov IB, Tkachuk VA. Stimulation of non-selective cation channels providing Ca2+ influx into platelets by platelet-activating factor and other aggregation inducers.Eur J Biochem 1991;198:267–273.PubMedGoogle Scholar
  20. 20.
    Hallam TJ, Rink TJ. Agonists stimulate divalent cation channels in the plasma membrane of human platelets.FEBS Lett 1985;186:175–179.PubMedGoogle Scholar
  21. 21.
    Sage SO, Rink TJ. Effects of ionic substitution on [Ca2+]1 rises evoked by thrombin and PAF in human platelets.Eur J Pharmacol 1986;128:99–107.PubMedGoogle Scholar
  22. 22.
    Davies TA, Drotts DL, Weil GJ, Simons ER. Cytoplasmic Ca2+ is necessary for thrombin-induced platelet activation.J Biol Chem 1989;264:19600–19606.PubMedGoogle Scholar
  23. 23.
    MacDougall SL, Grinstein S, Gelfand EW. Detection of ligand-activated conductive Ca2+ channels in human B lymphocytes.Cell 1988;54:229–234.PubMedGoogle Scholar
  24. 24.
    Tsien RY. New calcium indicators and buffers with high selectivity against magnesium and protons: Design, synthesis, and properties of prototype structures.Biochemistry 1980;19:2396–2404.PubMedGoogle Scholar
  25. 25.
    Motulsky HJ, Snavely MD, Hughes RJ, Insel PA. Interaction of verapamil and other calcium channel blockers with alpha 1- and alpha 2-adrenergic receptors.Circ Res 1983;52:226–231.PubMedGoogle Scholar
  26. 26.
    Schmitt R, Kleinbloesem CH, Belz GG. Hemodynamic and humoral effects of the novel calcium antoagonist Ro 40-5967 in patients with hypertension.Clin Pharmacol Ther 1992;52:314–323.PubMedGoogle Scholar
  27. 27.
    Portegies MCM, Schmitt R, Kraaij CJ, et al. Lack of negative inotropic effects of the new calcium antagonist Ro 40-5967 in patients with stable angina pectoris.J Cardiovasc Pharmacol 1991;18:746–751.PubMedGoogle Scholar

Copyright information

© Kluwer Academic Publishers 1995

Authors and Affiliations

  • Martina Hahn
    • 1
  • Vsevolod A. Tkachuk
    • 2
  • Valery N. Bochkov
    • 2
  • Ivan B. Cheglakov
    • 2
  • Jean-Paul Clozel
    • 1
  1. 1.PRPV, 70/411, Preclinical Research DepartmentF. Hoffmann-La Roche Ltd.BaselSwitzerland
  2. 2.Institute of Experimental CardiologyCardiology Research CentreMoscowRussia

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