Summary
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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.
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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.
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3.
Analysis of the saturation kinetics of 3H-5-HT uptake using Hill's equation yielded K m, V max and n H 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.
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4.
The n H 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 K m) 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.
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5.
Imipramine inhibited 3H-5-HT uptake by increasing the K m for 3H-5-HT without changing V max. The K i for this interaction was 18 nmol l−1.
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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 K i for imipramine and the concentration of plasma protein was linear; extrapolation to protein-free medium gave a K i value of 14.8 nmol l−1.
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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 n H and decreasing K m. Finally, imipramine acts as a purely competitive inhibitor, the potency of which being greatly affected by the presence of plasma protein.
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This study was supported by the Deutsche Forschungsgemeinschaft (Gr 490/6 and SFB 176)
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Wölfel, R., Böhm, W., Halbrügge, T. et al. On the 5-hydroxytryptamine transport across the plasma membrane of rabbit platelets and its inhibition by imipramine. Naunyn-Schmiedeberg's Arch Pharmacol 338, 1–8 (1988). https://doi.org/10.1007/BF00168804
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DOI: https://doi.org/10.1007/BF00168804