engleski

Platelet serotonin transporter : ex vivo monitoring of kinetic parameters in the individual rat

We have developed a method permitting full kinetic measurement of serotonin (5HT) transporter on rat platelets without the need for sacrifying the animal, and so the individual monitoring of this parameter over time. Blood sample of 1 mL was obtained by jugular venipuncture under light ether narcosis. Platelet rich plasma was prepared by centrifugation and quntitatively separated. After detailed testing of relevant methodological parameters the measurements were performed under following conditions: 50 mL aliquots of platelet rich plasma were incubated with 14C-5HT in 6 concentrations (0.07-1.00 mM, final) during 30 seconds at 37°C. The uptake was terminated by vacuum filtration at 550 mmHg. Blank values were obtained by repeating the procedure at 0°C. Values obtained on a sample of 31 animals were 2.86 ą 0.41 nmol 5HT/mg platelet protein/min for Vmax and 0.160 ą 0.024 mM for Km (MąSD). The described method enabled the performance of specific (patho)physiological studies on 5HT transporter ; furthermore, it permitted studies on genetic background for this parameter using selective breeding of animals for this trait. We have developed a method permitting full kinetic measurement of serotonin (5HT) transporter on rat platelets without the need for sacrifying the animal, and so the individual monitoring of this parameter over time. Blood sample of 1 mL was obtained by jugular venipuncture under light ether narcosis. Platelet rich plasma was prepared by centrifugation and quntitatively separated. After detailed testing of relevant methodological parameters the measurements were performed under following conditions: 50 mL aliquots of platelet rich plasma were incubated with 14C-5HT in 6 concentrations (0.07-1.00 mM, final) during 30 seconds at 37°C. The uptake was terminated by vacuum filtration at 550 mmHg. Blank values were obtained by repeating the procedure at 0°C. Values obtained on a sample of 31 animals were 2.86 ą 0.41 nmol 5HT/mg platelet protein/min for Vmax and 0.160 ą 0.024 mM for Km (MąSD). The described method enabled the performance of specific (patho)physiological studies on 5HT transporter ; furthermore, it permitted studies on genetic background for this parameter using selective breeding of animals for this trait.

5HT transporter; uptake kinetics; rat platelets

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