Abstract
Regulation of neutral amino acid transport was studied using isolated plasma membrane vesicles derived from the bovine blood-brain barrier. Neutral amino acids cross the blood-brain barrier by facilitative transport system L1, which may allow both desirable and undesirable amino acids to enter the brain. The sodium-dependent amino acid systems A and Bo,+ are located exclusively on abluminal membranes, in a position to pump unwanted amino acids out. gamma-Glutamyl transpeptidase, the first enzyme of the gamma-glutamyl cycle, is an integral protein of the luminal membrane of the blood-brain barrier. We demonstrate that oxoproline, an intracellular product of the gamma-glutamyl cycle, stimulates the sodium-dependent systems A and Bo,+ by 70 and 20%, respectively. Study of system A showed that 2 mM oxoproline increased the affinity for its specific substrate N-methylaminoisobutyrate by 50%. This relationship between the activity of the gamma-glutamyl cycle and system A transport may provide a short term regulatory mechanism by which the entry of potentially deleterious amino acids (i.e. neurotransmitters or their precursors) may be retarded and their removal from brain accelerated.
Highlights
The endothelial cells of cerebral capillaries are joined by tight junctions forming the blood-brain barrier (BBB).1 hydrophilic nutrients, such as amino acids, require the presence of carriers in the respective luminal and abluminal membranes to reach the brain
This overlap in transport activity coupled with the asymmetrical distribution of the amino acid transporters at the BBB provides a potential means for the regulation of amino acid delivery to the brain
1) Oxoproline accelerated the initial rate of substrate transport by the two sodiumdependent amino acid carriers known to be at the abluminal membrane of the BBB. 2) System A, which was stimulated to the greatest degree, manifested an increased affinity for its substrate
Summary
Materials—L-[ring-2,6-3H]Phenylalanine (46 Ci/mmol), N-[1-14C](methylamino)isobutyric acid (MeAIB, 56.3 mCi/mmol), L-[14C(U)]alanine (150.9 mCi/mmol), and [14C(U)]sucrose (475 mCi/mmol) were purchased from DuPont NEN. In addition to system A, there is another sodium-dependent transport system in abluminal membranes of the BBB (2). This system, known as Bo,ϩ, transports neutral and basic amino acids but has no affinity for MeAIB (30). To determine the transport activity of system Bo,ϩ, the uptake of alanine, a substrate of both systems A and Bo,ϩ, was measured in the presence of 5 mM MeAIB. This concentration is sufficient to block 90% of the activity associated with system A. A best fit analysis was employed to determine the kinetic parameters using the Michaelis-Menten equation and included a term for nonspecific diffusion, (Vmax*[S]/Km ϩ [S]) ϩ Kd[S]
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