Skip to main content

Binding of3H-desipramine to the neuronal noradrenaline carrier of rat phaeochromocytoma cells (PC-12 cells)

Naunyn-Schmiedeberg's Archives of Pharmacology volume 334, pages 403–411 (1986)Cite this article

Summary

The specific (i.e., nisoxetine-sensitive) binding of3H-desipramine was studied in purified plasma membranes of PC-12 cells (rat phaeochromocytoma cells).3H-desipramine bound reversibly and with high affinity (K D=4.5 nmol/l) to a single, non-interacting site (Hill coefficient=1.04); the maximal number of binding sites (B max) was 19.6 pmol/mg protein.

Like the uptake of noradrenaline (by uptake1), the binding of3H-desipramine was dependent on both sodium and chloride. The stimulation of binding by chloride and sodium was characterized by a Hill coefficient of about 1 and 2, respectively. Both, chloride and sodium, slowed the rate of dissociation of bound3H-desipramine. Increasing concentrations of sodium decreased theK D of3H-desipramine binding without altering theB max.

The binding of3H-desipramine was inhibited by tricyclic antidepressants and other noradrenaline uptake blockers. There was a highly significant correlation between the potencies of a series of drugs for the inhibition of3H-desipramine binding and for the inhibition of3H-noradrenanne uptake into intact PC-12 cells. Both, binding of3H-desipramine and uptake of3H-noradrenaline, were stereoselectively inhibited by the enantiomers of cocaine and oxaprotiline. However, for most of the substrates of uptake1 the IC50 for inhibition of3H-desipramine binding was much higher than that for inhibition of3H-noradrenaline uptake. Nevertheless, noradrenaline competitively inhibited3H-desipramine binding and unmasked dissociation of bound3H-desipramine. Thus,3H-desipramine probably binds to the substrate recognition site.

From theB max of3H-desipramine binding to PC-12 membranes and from theV max of3H-noradrenaline uptake into PC-12 cells, a duration of about 400 ms for the transport cycle for a single noradrenaline molecule was calculated. In addition, from theB max the maximum number of3H-desipramine binding sites (carriers) for a single PC-12 cell was calculated to be 55,000.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

43,34 €

Price includes VAT (Finland)
Tax calculation will be finalised during checkout.

References

  • Bönisch H (1984) The transport of (+)amphetamine by the neuronal noradrenaline carrier. Naunyn-Schmiedeberg's Arch Pharmacol 327:267–272

    Google Scholar 

  • Bönisch H, Harder R, Friedrich U, Schömig E (1984) Rat pheochromocytoma cells (PC-12 cells) as a model to study the transport of noradrenaline (NA) and the binding of desipramine (DMI). IUPHAR 9th International Congress of Pharmacology, London. Abstract 1212P

  • Briggs GE, Haldane JBS (1925) A note on the kinetics of enzyme action. Biochem J 19:338–339

    Google Scholar 

  • Christensen HN (1975) Biological transport, 2nd edn. WA Benjamin Inc, London Amsterdam

    Google Scholar 

  • DePaulis T, Kelder D, Ross SB (1978) On the topology of the norepinephrine transport carrier in rat hypothalamus. The site of action of tricyclic uptake inhibitors. Mol Pharmacol 14:596–606

    Google Scholar 

  • Friedrich U, Bönisch H (1986) The neuronal transport system of PC-12 cells: Kinetic analysis of the interaction between noradrenaline, Na+ and Cl− in transport. Naunyn-Schmiedeberg's Arch Pharmacol 333:246–252

    Google Scholar 

  • Graefe K-H (1976) Methodology of catecholamine transport studies: definition of terms. In: Paton DM (ed) The mechanism of neuronal and extraneuronal transport of catecholamines. Raven Press, New York, pp 7–35

    Google Scholar 

  • Graefe K-H, Bönisch H, Keller B (1978) Saturation kinetics of the adrenergic neurone uptake system in the perfused rabbit heart. A new method for the determination of initial rates of amine uptake. Naunyn-Schmiedeberg's Arch Pharmacol 302:263–273

    Google Scholar 

  • Greene LA, Rein G (1977) Release, storage and uptake of catecholamines by a clonal cell line of nerve growth factor (NGF) responsive pheochromocytoma cells. Brain Res 129:247–263

    Google Scholar 

  • Greene LA, Tischler AS (1982) PC-12 pheochromocytoma cultures in neurobiological research. Adv Cell Neurobiol 3:373–414

    Google Scholar 

  • Harder R, Bönisch H (1984) Large scale preparation of plasma membrane vesicles from PC-12 pheochromocytoma cells and their use in noradrenaline transport studies. Biochim Biophys Acta 775:95–104

    Google Scholar 

  • Iversen LL (1967) The uptake and storage of noradrenaline in sympathetic nerves. Cambridge University Press, Cambridge

    Google Scholar 

  • Keller B, Graefe K-H (1979) The inhibitory effect of some monovalent cations on the stimulation by Na+ of the neuronal uptake of noradrenaline. Naunyn-Schmiedeberg's Arch Pharmacol 309:89–97

    Google Scholar 

  • Langer SZ, Tahraoui L, Raisman R, Arbilla S, Najar M, Dedek J (1984)3H-Desipramine labels a site associated with the neuronal uptake of noradrenaline in the peripheral and central nervous system. In: Fleming WW, Graefe K-H, Langer SZ, Weiner N (eds) Neuronal and extraneuronal events in autonomic pharmacology. Raven Press, New York, pp 37–49

    Google Scholar 

  • Lee CM, Snyder SH (1981) Norepinephrine neuronal uptake binding sites in rat brain menbranes labeled with (3H)desipramine. Proc Natl Acad Sci USA 78:5250–5254

    Google Scholar 

  • Lee CM, Javitch JA, Snyder SH (1982) Characterization of (3H)desipramine binding associated with neuronal norepinephrine uptake sites in rat brain membranes. J Neurosci 2:1515–1525

    Google Scholar 

  • Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275

    Google Scholar 

  • Maxwell RA, Keenan PD, Chaplin E, Roth B, Eckardt SB (1969) Molecular features affecting the potency of tricyclic antidepressants and structurally related compounds as inhibitors of the uptake of tritiated norepinephrine by rabbit aortic strips. J Pharmacol Exp Ther 166:320–329

    Google Scholar 

  • Maxwell RA, Ferris RM, Burscu JE (1976) Structural requirements for inhibition of noradrenaline uptake by phenylethylamine derivatives, desipramine, cocaine, and other compounds. In: Paton DM (ed) The mechanism of neuronal and extraneuronal transport of catecholamines. Raven Press, New York, pp 95–153

    Google Scholar 

  • Michaelis L, Menten ML (1913) Die Kinetik der Invertinwirkung. Biochem Z 49:333–369

    Google Scholar 

  • Ohnishi ST, Barr JK (1978) A simplified method of quantitating protein using the biuret and phenol reagents. Anal Biochem 86:193–200

    Google Scholar 

  • Raisman R, Sette M, Pimoule C, Briley M, Langer SZ (1982) High-affinity3H-desipramine binding in the peripheral and central nervous system: a specific site associated with the neuronal uptake of noradrenaline. Eur J Pharmacol 78:345–352

    Google Scholar 

  • Rehavi M, Skolnick P, Brownstein MJ, Paul SM (1982) High-affinity binding of (3H)desipramine to rat brain: a presynaptic marker for noradrenergic uptake sites. J Neurochem 38:889–895

    Google Scholar 

  • Rudnick G, Talvenheimo J, Fishkes H, Nelson PJ (1983) Sodium requirements for serotonin transport and imipramine binding. Psychopharmacol Bull 19:545–549

    Google Scholar 

  • Sammet S, Graefe K-H (1979) Kinetic analysis of the interaction between noradrenaline and Na+ in neuronal uptake: kinetic evidence for co-transport. Naunyn-Schmiedeberg's Arch Pharmacol 309:99–107

    Google Scholar 

  • Sanchez-Armass S, Orrego F (1977) A major role of chloride in3H-noradrenaline transport by rat heart adrenergic nerves. Life Sci 20:1829–1838

    Google Scholar 

  • Segel IH (1975) Enzyme kinetics. Behaviour and analysis of rapid equilibrium and steady-state enzyme systems. John Wiley & Sons, New York London Sydney Toronto

    Google Scholar 

  • Ungell A-L, Bönisch H, Graefe K-H (1986) Choline+: a substrate of the neuronal noradrenaline carrier in the rat vas deferens. Naunyn-Schmiedeberg's Arch Pharmacol (in press)

  • Waldmeier PC, Baumann PA, Hauser K, Maitre L, Storini A (1982) Oxaprotiline, a noradrenaline uptake inhibitor with an active and an inactive enantiomer. Biochem Pharmacol 31:2169–2176

    Google Scholar 

  • Wilbrandt W, Rosenberg T (1961) The concept of carrier transport and its corollaries in pharmacology. Pharmacol Rev 13:109–183

    Google Scholar 

  • Williams LT, Lefkowitz RJ (1978) Receptor binding studies in adrenergic pharmacology. Raven Press, New York, pp 32–35

    Google Scholar 

Download references

Author information

Affiliations

  1. Institut für Pharmakologie und Toxikologie der Universität Würzburg, Versbacher-Strasse 9, D-8700, Würzburg, Federal Republic of Germany

    H. Bönisch & R. Harder

Authors
  1. H. Bönisch
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. R. Harder
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

This study was supported by the Deutsche Forschungsgemeinschaft (Bo 521 and SFB 176)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Bönisch, H., Harder, R. Binding of3H-desipramine to the neuronal noradrenaline carrier of rat phaeochromocytoma cells (PC-12 cells). Naunyn-Schmiedeberg's Arch. Pharmacol. 334, 403–411 (1986). https://doi.org/10.1007/BF00569378

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00569378

Key words

  • 3H-desipramine
  • Neuronal noradrenaline carrier
  • PC-12 cells