Kinetics of Neutral Amino Acid Transport Across the Blood‐Brain Barrier

Abstract

Neutral amino acid (NAA) transport across the blood-brain barrier was examined in pentobarbital-anesthetized rats with an in situ brain perfusion technique. Fourteen of 16 plasma NAAs showed measurable affinity for the cerebrovascular NAA transport system. Values of the transport constants (Vmax, Km, KD) were determined for seven large NAAs from saturation studies, whereas Km values for five small NAAs were estimated from inhibition studies. These data, together with our previous work, provide a complete set of constants for prediction of NAA influx from plasma. Among the NAAs, Vmax varied at least fivefold and Km varied approximately 700 fold. The apparent affinity (1/Km) of each NAA was related linearly (r = 0.910) to the octanol/water partition coefficient, a measure of NAA side-chain hydrophobicity. Predicted influx values from transport constants and average plasma concentrations agree well with values measured using plasma perfusate. These results provide accurate new estimates of the kinetic constants that determine NAA transport across the blood-brain barrier. Furthermore, they suggest that affinity of a L-alpha-amino acid for the transport system is determined primarily by side-chain hydrophobicity.

Characterization of Cerebral Glutamine Uptake from Blood in the Mouse Brain: Implications for Metabolic Modeling of 13C NMR Data

“…The rate of glutamine influx (V Gln(in) , 0.036±0.002 mmol/g per minute), as measured under near constant and saturating levels of plasma glutamine (1.8 mmol/L), is similar to the blood-to-brain maximum transport rate, V max , of 0.043 mmol/g per minute reported in anesthetized rats in vivo by Smith et al 23 Values of V max measured from the uptake of glutamine incubated in vitro using brain slices vary substantially. For example, the V max reported by Benjamin et al 24 for nonstimulated rat cortical slices maintained at 371C of 0.2 and 0.5 mmol/g over 12 minutes (corresponding to 0.017 and 0.042 mmol/g per minute, respectively) from uptake of L-[U- 14 C]glutamine over a range of glutamine concentrations between 67 mmol/L and 2 mmol/L is from 6 to 13 times lower than the V max of 0.23 mmol/g per minute reported by Balcar and Johnston 25 for slices of cerebral gray matter at 251C for a much lower range of medium glutamine levels (5 to 60 mmol/L).…”

“…This is likely an upper limit considering that glutamine in plasma would compete with other nonpolar amino acids for transport into the brain. 23 Blood-to-Brain Glutamine Influx The rate of glutamine influx from blood, V Gln(in) , was determined by modeling the time courses of 13 C enrichment of brain glutamine-C4 and glutamate-C4,C3 from [U- 13 C 5 ]glutamine resulting in a quasi-constant plasma glutamine level of B1.8 mmol/L, which was above the concentration at which transport was saturated. This approach treated V Gln(in) as an independent iterated parameter ( Figure 1A).…”

“…This value is considerably higher than Table 1. Concentrations (mmol/L) and 13 C enrichments (%) of plasma and brain amino acids during intravenous [U- 13 Glutamine transport in mouse brain P Bagga et al previously reported rates of glutamine influx for physiologic plasma glutamine concentrations (0.42 to 0.48 mmol/L) of 0.0049 mmol/g per minute 26 or 0.0041 mmol/g per minute in parietal cortex 23 as measured with L-[ 14 C]glutamine and in situ carotid artery perfusion. However, under physiologic condition, several amino acids present in blood may compete with glutamine for transport, effectively raising the apparent plasma concentration required for half maximal transport, K m ', and reducing its influx as much as 22-fold.…”

“…However, under physiologic condition, several amino acids present in blood may compete with glutamine for transport, effectively raising the apparent plasma concentration required for half maximal transport, K m ', and reducing its influx as much as 22-fold. 23 Because our study was conducted with elevated and saturating blood glutamine concentrations, effectively reducing the effects of other competing amino acids, our estimate of V Gln(in) for physiologic blood glutamine level likely reflects an upper limit. Compared with the in vivo estimates, reported maximum rates of glutamine transport in cultured neurons and astrocytes are much higher (e.g., V max of 0.7 to 5.0 mmol/g per minute assuming 100 mg protein/g), [27][28][29] suggesting that the lower rates measured in vivo reflect mainly transport through the blood-brain barrier.…”

Differential axial localization along the mouse brain vascular tree of luminal sodium-dependent glutamine transporters Snat1 and Snat3

“…Both functional transport and the expression of a marker (g-glutaminyl transferase) for luminal Na + -dependent AA transport has been detected. In addition, early in vivo studies by Oldendorf and others (reviewed in the study by Smith et al (1987)) measured a low but significant brain uptake for glutamine that was attributed to a neutral AA carrier system. Later, Ennis et al (1998) showed a Na + -dependent, pH-sensitive glutamine luminal transport that was fully inhibited by histidine but not by cystine (system A or ASC inhibitors) or 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) (system L inhibitor) and therefore identified as system N. In addition to our present report showing Snat3 protein expression, our previous study additionally found a robust and regulated gene Snat1 is localized on the luminal membrane of brain vasculature, but not on BBB capillaries.…”