Naslov
Methodological considerations on kinetic measurements of serotonin transport into rat platelets

Autori
Frobe, Ana ; Čičin-Šain, Lipa ; Reiner, Elsa ; Banović, Miroslav ; Jernej, Branimir

Izvornik
Periodicum Biologorum (0031-5362) 97 (1995), 3; 217-222

Vrsta, podvrsta i kategorija rada
Radovi u časopisima, članak, znanstveni

Ključne riječi
serotonin uptake; rat platelets; 5HT transporter

Sažetak
Background and purpose: Blood platelets represent an attractive peripheral model in neuropsychopharmacology owing to serotonin transmembrane uptake mechanism - target for different psychoactive drugs, mainly antidepressants. The transport kinetics of serotonin (5-hydroxytryptamine, 5HT) through the plasma membrane is interpreted by the Michaelis-Menten equation and results expressed in terms of Km and Vmax. Published values of kinetic parameters for the 5HT transport into rat platelets vary considerably. The purpose of this paper was to evaluate possible reasons of these varioations, with special emphasis given on methodology of kinetic measurements. Material and methods: Platelet rich plasma was separated from anticoagulated blood samples (1 mL) obtained by jugular venipuncture from ether-anesthetized rats. Kinetic measurements were performed at 37 C by incubating various number of rat platelets with several 14C-5HT concentrations during different time intervals. The uptake was terminated by rapid cooling of incubating mixture and separation of platelets by filtration at different vacuum settings. Platelet rich plasma was stored at room temperature for different time periods prior to kinetic measurements. Serotonin, taken up by platelets, was quantified as beta counts of 14C in liquid scintillator. Calculation of kinetic parameters, Km and Vmax, was performed using Eadie-Hofstee plot. Results: Based on the results of methodological studies, experimental conditions which, by our experience, assure reliable and reproducible kinetic measurements of 5HT tranasport into rat platelets were defined as follows: 1) concentration of platelets in incubating mixture: 25-30×10^6/uL of incubating medium ; b) concentrations of substrate (14C-5HT) in incubating medium: 0.05, 0.07, 0.10, 0.15, 0.25, 0.50 and 1.00 uM ; c) uptake interval: 30 seconds ; d) vacuum setting for filtration: 600 mmHg ; e) recommended storage of platelet suspension prior to kinetic determinations: up to three hours at room temperature. Values of kinetic parameters for serotonin uptake into rat platelets obtained by this methodology were: Vmax = 225 +/- 37 pmol 5HT ×10^8 plt/min and Km = 0.161 +/- 0.023 uM (M +/- SD, N = 27). Conclusion: Based on thorough methodological studies the procedure for reliable measurement of kinetic parameters of serotonin uptake into rat platelets was described with critical review of available literature in the field. Such methodology can serve as a basis for further development of the experimental model for ex vivo monitoring of platelet serotonin uptake in rats.

Izvorni jezik
Engleski

Znanstvena područja
Biologija, Temeljne medicinske znanosti

POVEZANOST RADA