On the 5-hydroxytryptamine transport across the plasma membrane of rabbit platelets and its inhibition by imipramine

  • R. Wölfel
  • W. Böhm
  • T. Halbrügge
  • H. Bönisch
  • K. H. Graefe
Article
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Summary

  1. 1.

    The carrier-mediated uptake of labelled 5-hydroxytryptamine (3H-5-HT) in rabbit platelets (defined as the difference between uptake observed in the absence and presence of 10 μmol l−1 imipramine) was studied after inhibition of monoamine oxidase and after a 1:13 dilution of the platelet-rich plasma (PRP) with Tris-containing buffer.

     
  2. 2.

    Irrespective of whether the rabbits were pretreated with reserpine or not, initial rates of 3H-5-HT uptake were maintained for at least 15 s.

     
  3. 3.

    Analysis of the saturation kinetics of 3H-5-HT uptake using Hill's equation yielded Km, Vmax and nH values of 130 nmol l−1, 116 pmol 108 platelets−1 min−1 and 1.40, respectively. Pretreatment of the animals with reserpine did not affect any of these kinetic parameters, but depleted more than 99% of the platelets' 5-HT stores.

     
  4. 4.

    The nH value remained greater than unity when the duration of incubation with 3H-5-HT was extended from 15 to 30 s and when the uptake of 3H-5-HT was inhibited by the presence of imipramine (10–40 nmol l−1), However, it was reduced to unity (with a consequential increase in Km) when 300 nmol l−1 ketanserin was present. This concentration of ketanserin did not affect 3H-5-HT uptake at substrate concentrations far below Km.

     
  5. 5.

    Imipramine inhibited 3H-5-HT uptake by increasing the Km for 3H-5-HT without changing Vmax. The Ki for this interaction was 18 nmol l−1.

     
  6. 6.

    When the fractional amount of PRP in the final incubation mixture was gradually increased from 1/13 to 1/2, there was a progressive parallel shift of the concentration-effect curve for imipramine to the right. The relationship between the log Ki for imipramine and the concentration of plasma protein was linear; extrapolation to protein-free medium gave a Ki value of 14.8 nmol l−1.

     
  7. 7.

    The results indicate that true initial rates of 3H-5-HT uptake were determined, since the pretreatment with reserpine did not affect any of the kinetic parameters for uptake. The shape change reaction of the platelets elicited by 5-HT appears to modulate the saturation kinetics of 3H-5-HT uptake by increasing nH and decreasing Km. Finally, imipramine acts as a purely competitive inhibitor, the potency of which being greatly affected by the presence of plasma protein.

     

Key words

5-HT uptake Reserpine Imipramine Platelets Plasma protein 
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References

  1. Ahtee L, Boullin DJ, Saarnivaara L, Paasonen MK (1974) The inhibition of the uptake of monoamines into platelets by phenothiazines and other drugs. In: Forrest IE, Carr CJ, Usdin E (eds) The phenothiazines and structurally related drugs. Raven Press, New York, pp 379–388Google Scholar
  2. Akera T, Brody MB (1987) The role of Na+,K+-ATPase in the inotropic action of digitalis. Pharmacol Rev 29:187–220Google Scholar
  3. Born GVR, Juengjaroen K, Michael F (1972) Relative activities on and uptake by human blood platelets of 5-hydroxytryptamine and several analogues. Brit J Pharmacol 44:117–139Google Scholar
  4. Briley M (1985) Imipramine binding: its relationship with serotonin uptake and depression. In: Green AR (ed) Neuropharmacology of serotonin. Oxford University Press, Oxford New York Toronto Melbourne, pp 50–78Google Scholar
  5. Briley M, Langer SZ, Sette M (1981) Allosteric interaction between the 3H-imipramine binding site and the serotonin uptake mechanism. Brit J Pharmacol 74:817P–818PGoogle Scholar
  6. Cheng YC, Prusoff WH (1973) Relationship between the inhibition constant (K i) and the concentration of inhibitor which causes 50 percent inhibition (I50) of an enzymic reaction. Biochem Pharmacol 22:3099–3108CrossRefPubMedGoogle Scholar
  7. De Clerk F, David J-L, Janssen PAJ (1982) Inhibition of 5-hydroxytryptamine-induced and -amplified human platelet aggregation by ketanserin (R 41468), a selective 5-HT2-receptor antagonist. Agents and Actions 12:388–397Google Scholar
  8. Da Prada M, Picotti GB (1986) Content and subcellular localization of catecholamines and 5-hydroxytryptamine in human and animal blood platelets: monoamine distribution between platelets and plasma. Brit J Pharmacol 65:653–662Google Scholar
  9. Erne P, Pletscher A (1985) Rapid intracellular release of calcium in human platelets by stimulation of 5-HT2-receptors. Brit J Pharmacol 84:545–549Google Scholar
  10. Frojmovic MM, Milton JG (1982) Human platelet size, shape, and related functions in health and disease. Physiol Rev 62:185–261Google Scholar
  11. Given MB, Longenecker GL (1985) Characteristics of serotonin uptake and release by platelets. In: Longenecker GL (ed) The platelets, physiology and pharmacology. Academic Press, Orlando San Diego New York London Toronto Montreal Sydney Tokyo, pp 463–479Google Scholar
  12. Janssen PAJ (1985) Pharmacology of potent and selective S2-serotonergic antagonists. J Cardiovasc Pharmacol 7, Suppl 7:S2-S11Google Scholar
  13. Javaid JI, Hendricks K, Davis JM (1983) α1-Acid glycoprotein involvement in high-affinity binding of tricyclic antidepressants to human plasma. Biochem Pharmacol 32:1149–1153Google Scholar
  14. Keyes SR, Rudnick G (1982) Coupling of transmembrane proton gradients to platelet serotonin transport. J Biol Chem 257: 1172–1176Google Scholar
  15. Lampugnani MG, De Gaetano G (1982) Comparative effects of ketanserin, a novel serotonergic receptor antagonist, on 5-HT-induced shape change and 5-HT uptake in rat and human platelets. Biochem Pharmacol 31:3000–3002Google Scholar
  16. Le Fur G, Uzan A (1977) Effects of 4-(3-indolyl-alkyl)piperidine derivatives on uptake and release of noradrenaline, dopamine and 5-hydroxytryptamine in rat brain synaptosomes, rat heart and human blood platelets. Biochem Pharmacol 26:479–503Google Scholar
  17. Lingjaerde O (1979) Inhibitory effects of clomipramine and related drugs on serotonin uptake in platelets: more complicated than previously thought. Psychopharmacology 61:245–249Google Scholar
  18. Meyerson LR, Ieni JR, Wennogle LP (1987) Allosteric interaction between the site labeled by 3H-imipramine and the serotonin transporter in human platelets. J Neurochem 48:560–565Google Scholar
  19. Müller WE, Stillbauer AE, El-Gamal S (1983) Psychotropic drug competition for 3H-imipramine binding further indicates the presence of only one high affinity binding site on human α1-acid glycoprotein. J Pharm Pharmacol 35:684–686Google Scholar
  20. Peterson PL (1977) A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal Biochem 83:346–356Google Scholar
  21. Pletscher A (1968) Metabolism, transfer and storage of 5-hydroxytryptamine in blood platelets. Brit J Pharmacol 32:1–16Google Scholar
  22. Rehavi M, Ittah Y, Skolnick P, Rice KC, Paul SM (1982) Nitroimipramine-synthesis and pharmacological effects of potent long-acting inhibitors of 3H-serotonin uptake and 3H-imipramine binding. Naunyn-Schmiedeberg's Arch Pharmacol 320:45–49Google Scholar
  23. Reimers H-J, Allen DJ, Cazenave J-P, Feuerstein IA, Mustard JF (1977) Serotonin transport and storage in rabbit blood platelets — the effects of reserpine and imipramine. Biochem Pharmacol 26:1645–1655Google Scholar
  24. Rudnick G (1977) Active transport of 5-hydroxytryptamine by plasma membrane vesicles isolated from human blood platelets. J Biol Chem 252:2170–2174Google Scholar
  25. Rudnick G, Fishkes H, Nelson J, Schuldiner S (1980) Evidence for two distinct serotonin transport systems in platelets. J Biol Chem 255:3638–3641Google Scholar
  26. Rudnick G, Talvenheimo J, Fishkes H, Nelson PJ (1983) Sodium ion requirements for serotonin transport and imipramine binding. Psychopharmacol Bull 19:545–549Google Scholar
  27. Segel IH (1975) Enzyme kinetics. Behaviour and analysis of rapid equilibrium and steady-state systems. John Wiley and Sons, New York London Sydney TorontoGoogle Scholar
  28. Segonzac A, Raisman R, Tateishi T, Schoemaker H, Hicks PE, Langer SZ (1985) Tryptamine, a substrate for the serotonin transporter in human platelets, modifies the dissociation kinetics of 3H-imipramine binding: possible allosteric interaction. J Neurochem 44:349–356Google Scholar
  29. Segonzac A, Tateishi T, Langer SZ (1984) Saturable uptake of 3H-tryptamine in rabbit platelets is inhibited by 5-hydroxytryptamine uptake blockers. Naunyn-Schmiedeberg's Arch Pharmacol 328:33–37Google Scholar
  30. Sette M, Briley MS, Langer SZ (1983) Complex inhibition of 3-Himipramine binding by serotonin and nontricyclic serotonin uptake blockers. J Neurochem 40:622–628Google Scholar
  31. Sneddon JM (1973) Blood platelets as a model for monoamine-containing neurones. Prog Neurobiol 1:151–198Google Scholar
  32. Snedecor GW, Cochran WG (1967) Statistical methods. Iowa State University Press, AmesGoogle Scholar
  33. Stacey RS (1961) Uptake of 5-hydroxytryptamine by platelets. Brit J Pharmacol 16:284–295Google Scholar
  34. Stahl SM (1985) Platelets as pharmacological models for the receptors and biochemistry of monoaminergic neurons. In: Longenecker GL (ed) The platelets, physiology and pharmacology. Academic Press, Orlando San Diego New York London Toronto Montreal Sydney Tokyo, pp 307–340Google Scholar
  35. Talvenheimo J, Nelson PJ, Rudnick G (1979) Mechanism of imipramine inhibition of platelet 5-hydroxytryptamine transport. J Biol Chem 254:4631–4635Google Scholar
  36. Tuomisto J (1974) A new modification for studying 5-HT uptake by blood platelets: a re-evaluation of tricyclic antidepressants as uptake inhibitors. J Pharm Pharmacol 26:92–100Google Scholar
  37. Wielosz M, Salmana M, De Gaetano G, Garattini S (1976) Uptake of 14C-5-hydroxytryptamine by human and rat platelets and its pharmacological inhibition. Naunyn-Schmiedeberg's Arch Pharmacol 296:59–65Google Scholar
  38. Wölfel R, Böhm W, Halbrügge T, Graefe K-H (1987) On the serotonin (5-HT) carrier system in platelets. Naunyn-Schmiedeberg's Arch Pharmacol 335:R88Google Scholar
  39. Zavoico GB, Cragoe EJ, Feinstein MB (1986) Regulation of intracellular pH in human platelets. J Biol Chem 261: 13160–13167Google Scholar

Copyright information

© Springer-Verlag 1988

Authors and Affiliations

  • R. Wölfel
    • 1
  • W. Böhm
    • 1
  • T. Halbrügge
    • 1
  • H. Bönisch
    • 1
  • K. H. Graefe
    • 1
  1. 1.Institut für Pharmakologie und Toxikologie der Universität WürzburgWürzburgFederal Republic of Germany

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