THE HUMAN PLATELET AS A MODEL OF THE GLUTAMATERGIC SYNAPSE OF THE CENTRAL NERVOUS SYSTEM
Many aspects of brain function are impossible to study in the human brain because tissue samples are difficult to obtain. It would be extremely helpful, therefore, if changes occuring in brain tissue could be monitored, even in part, in other more easily accessible peripheral tissue. There is a rationale for such an idea because many cells share certain basic properties, such as membrane structure and mechanisms of ion transport. The human blood platelet seem to fulfill some of the requirements: it is easily accessible and shares a number of similar properties with brain synaptosomes. For instance, platelets accumulate amines and amino acids against a concentration gradient, and they possess amine storage vesicles along with enzymes (monoamine oxidase, amino acid transaminase) similar to those found in the brain. A platelet model for the study of glutamate uptake in the brain would provide an opportunity to study the role of glutamate uptake in Huntington's disease (HD), an autosomal dominant, neurodegenerative disease.^ A method was devised for isolating human platelets, free from erythrocyte, leukocyte, and plasma protein contamination. The optimum assay conditions for glutamate uptake were determined to be a 10 minute incubation at 37(DEGREES)C in Tris-citrate buffer, pH 6.5. No metabolism of glutamate or aspartate was observed during the 10 minute incubations. Glutamate uptake was rapidly stopped by either cooling the platelets to 0-4(DEGREES)C or by fixing the platelets in 1.5% phosphate buffered formaldehyde. Once uptake was stopped, no leakage of accumulated radiolabel was observed. Whole blood could be stored for 6 hours prior to platelet isolation without affecting glutamate uptake but once suspended in 0.32M sucrose, platelets must be used immediately.^ Human platelet glutamate uptake was time dependent and linear from 0-5 minutes. Platelet glutamate uptake was also markedly temperature- and Na('+) dependent. Uptake at 2.5(DEGREES)C was 8.66% of control (37(DEGREES)C) uptake. No uptake was observed at a Na('+) concentration of 15 mM, but uptake increased linearly from 15-120 mM Na('+). Ouabain (1x10('-3)M) inhibited glutamate uptake by 29.5%. Rat platelets also accumulated glutamate but by a process that was not saturable at 1 x 10('-3)M glutamate. Ouabain (1 x 10('-3)M) inhibited rat platelet accumulation of glutamate by 37.3%.^ The human platelet carrier for glutamate was observed to be specific for glutamate and aspartate. Dopamine, serotonin, taurine, and GABA, all compounds accumulated by human platelets, did not inhibit glutamate uptake at the concentrations examined. D-glutamic acid (5 x 10('-4)M) and kainic acid (5 x 10('-4)M) inhibited glutamate uptake by 24.8% and 12.3%, respectively. The glutamate analogues, threo-3-hydroxy-DL-aspartate and DL-aspartate-beta-hydroxamate had IC(,50) values of 1.4x10('-5)M and 2.3x10('-5)M, respectively, for glutamate uptake.^ The kinetics of glutamate and aspartate uptake were investigated in both normal and HD platelets. The uptake mechanism consisted of both high and low affinity components. Control platelets had high affinity K(,m) values of 3.1 (mu)M and 2.2 (mu)M, and low affinity K(,m) values of 88.2 (mu)M and 149.0 (mu)M for glutamate and aspartate uptake, respectively. The kinetic constants for glutamate and aspartate uptake in HD platelets were not significantly different from control values. ^
MANGANO, RICHARD MICHAEL, "THE HUMAN PLATELET AS A MODEL OF THE GLUTAMATERGIC SYNAPSE OF THE CENTRAL NERVOUS SYSTEM" (1980). ETD Collection for Fordham University. AAI8020071.