L. A. KASATKINA1,2 , T. A. BORISOVA1
DIRECT AND REVERSED FUNCTION OF Na+- DEPENDENT GLUTAMATE TRANSPORTERS IN BLOOD PLATELETS
1Palladin Institute of Biochemistry, NAS of Ukraine, Kyiv;
2 Taras Shevchenko National University of Kyiv, Biological Faculty, Kyiv
Blood platelets contain Na+-dependent glutamate transporters similar to those located in the plasma membrane of neurons. The aim of the research was to analyze comparatively direct and reversed transport of glutamate in rabbit blood platelets and rat brain nerve terminals (synaptosomes). Km value of the Na+-dependent L-[14C]glutamate uptake consisted of 36 ± 8 µM for platelets and 11 ± 2 µM for nerve terminals. Vmax was three orders lower for platelets (5.2 ± 1.3 pmol · min-1 · mg-1 of proteins) as compared with synaptosomes (12.5 ± 3.2 nmol · min-1 · mg-1 of proteins).
Applying the drug 4-aminopyridine (4-AP), which blocks voltage-dependent potassium conductance, and high-KCl, we have demonstrated dose-dependent depolarization of the plasma membrane of platelets that was registered as an increase in the fluorescence of the potential-sensitive fluorescent dye rhodamine 6G.
The initial velocity of L-[14C]glutamate uptake (10 µM) in platelets was decreased by 14 % during 2 mM 4-AP- and by 20 % during 35 mM KCl - evoked depolarization and was equal to 1.2 ± 0.09 pmol · min-1 · mg-1 protein in control, 1.02 ± 0.08 pmol · min-1 · mg-1 protein in the presence of 4-AP and 0.96 ± 0.08 pmol · min-1 · mg-1 protein in the presence of high-KCl. Confocal laser scanning microscopy and flow cytometry revealed that these changes in glutamate uptake were not a result of platelet aggregation/activation.
Depolarization of the synaptosomal plasma membrane with 4-AP and 35 mM KCl decreased the initial velocity of 10 µM L-[14C]glutamate uptake by 16 % and 50 %, respectively, that consisted of 3.0 ± 0.3 nmol · min-1 · mg-1 protein in control, 2.53 ± 0.2 nmol · min-1 · mg-1 for 4-AP and 1.52 ± 0.2 nmol · min-1 · mg-1 protein for high-KCl.
Glutamate transporter reversal during depolarization of the plasma membrane was also assessed comparatively in platelets and synaptosomes using glutamate dehydrogenase assay or applying L-[14C]glutamate. It was shown that in contrast to nerve terminals where Ca2+ - independent L-[14C]glutamate release was equal to 14 ± 2 % of total label, it was not registered in platelets.
Thus, depolarization of the plasma membrane of platelets resulted in a decrease in direct transport of glutamate but did not cause reverse of glutamate transporters. Weak glutamate uptake during depolarization of the plasma membrane might have considerable consequences for platelets per se (and thus for haemostatic system) and glutamate homeostasis in the CNS. This malfunction of glutamate transporters has to take place under: (1) the conditions of pseudohyperkalemia or hyperkalemia, i.e. activation and clotting of platelets, haemolysis, leucocytosis, acute renal failure, hypofunction of adrenal cortex, lack of aldosterone, stroke, trauma; (2) depolarization of the plasma membrane of platelets during their activation by ADP, thrombin, platelet-activating factor.