Increased brain uptake and brain to blood efflux transport of 14C-GABA in spontaneously hypertensive rats.

The brain uptake and brain to blood efflux transport of (14)C-GABA were studied in spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto (WKY) rats using 20 min bilateral in situ brain perfusion in rats anesthetized using urethane. The volume of distribution (Vd) of (14)C-GABA into cerebrospinal fluid (CSF) and brain regions (cortex, diencephalon, cerebellum, and brain stem) was significantly greater in SHR than in the corresponding regions in WKY rats (p<0.05). The estimated Vd value of (14)C-GABA in CSF of SHR was 3.4 fold greater than that in WKY. Also compared to WKY, the Vd of (14)C-GABA into cerebellum and cortex of SHR was 15.3 fold and 19.4 fold greater, respectively. Although the study of blood-brain barrier (BBB) integrity using (3)H-mannitol revealed increased paracellular permeability at the brain capillaries of SHR when compared to WKY rats, this was found to be only partially responsible for the increased (14)C-GABA uptake. The study of brain to blood efflux transport of (14)C-GABA (after loading of brain with (14)C-GABA by vascular perfusion) revealed that the half-time of elimination was significantly shorter in SHR (5.35+/-0.66 min) than in WKY rats (14.83+/-1.94 min), (p<0.001). HPLC analysis revealed that GABA concentrations in brain extracts and CSF of SHR were similar to those in WKY rats (p>0.05). The faster efflux in SHR might be, at least partially, responsible to compensate for increased uptake of this neurotransmitter and to preserve the protective function of BBB towards GABA. The protective function of the BCSFB towards GABA appears to be also preserved, since systemic infusion of GABA within a wide range of administered doses (0.004-5.00 mg/kg) produced an increase in GABA CSF concentration from around 0.5 microM to only 11 microM, and the obtained pattern of CSF GABA concentrations under these conditions did not differ between SHR and WKY rats, as revealed by HPLC.

Acidic amino acid clearance from CSF in the neonatal versus adult rat using ventriculo-cisternal perfusion.

The acidic amino acids aspartate and glutamate are excitatory neurotransmitters in the CNS. The clearance of this group of amino acids from CSF of adult and neonatal (7-day-old) rats was investigated. Ventriculo-cisternal perfusions with 14C-amino acids and 3H-dextran were carried out for up to 90 min. Uptake of the amino acids by the whole brain was measured, and the loss to blood was calculated. 3H-Dextran was included in the perfusate for measurement of CSF secretion rate. After 90-min perfusion, both aspartate and glutamate showed a similar uptake into the whole brain, and this did not change with age (p>0.05). However, clearance from CSF was greater in the adult, as was entry into blood from CSF. Addition of 5 mM excess unlabelled amino acid resulted in reduction in the brain uptake of both 14C-amino acids in the adult rat. In the neonate, addition of aspartate also reduced brain aspartate uptake, whereas addition of glutamate increased brain neonatal [14C]glutamate uptake. The rate of CSF secretion was significantly greater in the adult, 1.26+/-0.18 microl x min(-1) x g(-1), than in the neonate, 0.62+/-0.08 microl x min(-1) x g(-1), and the turnover of CSF was greater in adults (p<0.01). In summary, both aspartate and glutamate showed greater clearances from CSF in the adult than the neonate. This clearance was found to be by carrier-mediated mechanisms.

Acidic amino acid accumulation by rat choroid plexus during development.

Acidic amino acid accumulation by the choroid plexuses of the lateral ventricles was investigated using 1, 2, 3 week and adult (7-10 weeks old) rats. The accumulation from both blood and CSF sides of the choroid plexuses were investigated. The uptake from blood side was studied using the bilateral in situ brain perfusion, and time-dependent uptake profiles (2, 10, 20, and 30 min) of 14C-labelled aspartate, glutamate, and NMDA were measured. [3H]Mannitol was also included in perfusion fluid as a baseline for [14C]amino acid uptake into choroidal tissue. Uptake of [14C]aspartate and [14C]glutamate declined with age, while [14C]NMDA showed no significant uptake at any age. Twenty min [3H]mannitol uptake in the 1-week-old rat was significantly greater than the adult (P < 0.05). The K(m) for [14C]aspartate and [14C]glutamate obtained from multiple time uptake profiles also showed reduction with development but it was greater than that for mannitol. [14C]Aspartate declined from 69.8 +/- 21.1 microliters.min-1.g-1 in the neonate to 40.6 +/- 4.0 microliters.min-1.g-1 in the adult (P < 0.05), while glutamate showed a sharper decline from 78.9 +/- 24.2 microliters.min-1.g-1 to 17.7 +/- 5.4 microliters.min-1.g-1 (P < 0.01). Accumulation of 14C-labelled aspartate and glutamate by the choroid plexus from CSF side was also measured using ventriculo-cisternal perfusion. The accumulation in the adult was found to be 2-3 times greater than that in the neonatal rat (P < 0.05) for both amino acids. The uptake from either side was found to be saturable, stereospecific, not inhibited by neutral amino acid analogues, and shared by both aspartate and glutamate.

Changes in the kinetics of the acidic amino acid brain and CSF uptake during development in the rat.

Using a bilateral in situ brain perfusion technique, the rate of influx of the acidic amino acids, aspartate and glutamate, into both brain and CSF, were measured in the rat. The kinetic constants for uptake of these amino acids across the blood-brain and blood-CSF barriers in neonatal (1-week-old) and adult (7-10 weeks-old) rats were calculated; the half saturation constant (K(m)) at both barriers did not change with age, whereas the maximal transport (Vmax) at both barriers was greater in the younger age group, and reduced by more than 50% with maturity. The diffusion constant Kd at the blood-brain barrier was not different from zero at either age, although at the blood-CSF barrier there was some diffusion at both ages, which did not change with maturity. The entry of these amino acids into the neonatal brain shown in our previous study can be explained by a greater maximal transport in the neonates which, coupled with the elevated plasma amino acid concentrations of the young animal, would result in higher blood-to-brain and blood-to-CSF flux in the neonate.

The entry of acidic amino acids into brain and CSF during development, using in situ perfusion in the rat.

Previous studies using the rapid single pass blood to tissue uptake of substances by the capillaries of the blood-brain barrier, have failed to show significant uptake of acidic amino acids. However, by the use of a bilateral in situ brain perfusion in neonatal and adult rats, extending the perfusion time to 30 min, the carrier-mediated uptake of aspartate and glutamate into brain and CSF has been demonstrated. The ratios of 14C-acidic amino acids in the brain and CSF to that in perfusate were measured and represented as Rbrain and RCSF respectively, after 30 min, neonatal (1-week-old) Rbrain values for both amino acids were approximately twice that of adults, while neonatal RCSF for aspartate and glutamate were 3 to 5 times that of the adult. In contrast, there was no significant entry of NMDA into either compartment for both adults and neonates. The transfer coefficient, Kin into brain and CSF was also measured in relation to stages of development. In general the Kin values for brain and CSF for aspartate and glutamate were higher in the younger age groups than the adult group (1 week > 2 week > 3 week > or = adult). In 1- and 2-week-old rats entry into CSF appears to be higher than that of brain, whereas for adults entry into the brain tissue was dominant.

Permeability of the developing blood-brain barrier to 14C-mannitol using the rat in situ brain perfusion technique.

The brain penetration of 14C-mannitol was investigated using a bilateral in situ brain perfusion technique followed by capillary depletion analysis. This technique measures the uptake of slowly penetrating solutes in the absence of the systemic circulation, and separates accumulation in brain endothelial cells from uptake into brain parenchyma. Penetration of 14C-mannitol was linear up to 30 min in rats aged 1, 2, 3 weeks and in adults. The brain mannitol space was higher in 1-week-old neonatal rats compared with adults (P < 0.05) and was due to a greater initial volume of distribution (Vi) for mannitol in the neonates, and not due to an elevated transfer rate (K(in)). Thirty percent of mannitol in the neonatal brain was associated with the capillary containing fraction, whereas in the adult only 13% was found in this fraction. This suggests that the permeability of the blood-brain barrier to mannitol does not change significantly with development but that more mannitol is associated with endothelial cells in the neonate. An investigation of 14C-glycine uptake was also carried out, and unlike mannitol the K(in) was greater in the neonate compared to the adult suggesting an elevated rate of transfer for this amino acid into the neonatal rat brain.