Changes in kinetics of amino acid uptake at the ageing ovine blood-cerebrospinal fluid barrier.

Amino acids (AA) in brain are precisely controlled by blood-brain barriers, which undergo a host of changes in both morphology and function during ageing. The effect of these age-related changes on AA homeostasis in brain is not well described. This study investigated the kinetics of four AA (Leu, Phe, Ala and Lys) uptakes at young and old ovine choroid plexus (CP), the blood-cerebrospinal fluid (CSF) barrier (BCB), and measured AA concentrations in CSF and plasma samples. In old sheep, the weight of lateral CP increased, so did the ratio of CP/brain. The expansion of the CP is consistent with clinical observation of thicker leptomeninges in old age. AA concentrations in old CSF, plasma and their ratio were different from the young. Both V(max) and K(m) of Phe and Lys were significant higher compared to the young, indicating higher trans-stimulation in old BCB. Cross-competition and kinetic inhibition studies found the sensitivity and specificity of these transporters were impaired in old BCB. These changes may be the first signs of a compromised barrier system in ageing brain leading increased AA influx into the brain causing neurotoxicity.

Age-related changes in choroid plexus and blood-cerebrospinal fluid barrier function in the sheep.

Dysfunction of the choroid plexuses (CPs) and the blood-cerebrospinal fluid barrier (BCSFB) might contribute to age-related cognitive decline and neurodegenerative disease. We used the CPs from young (1-2 years), middle-aged (3-6 years) and old (7-10 years) sheep to explore effects of aging on various aspects of CP and BCSFB functions. Total protein in the cerebrospinal fluid (CSF) was significantly higher in old compared to young sheep and CSF secretion by the CP perfused in situ was significantly lower in both old and middle-aged when compared to young sheep, which correlated with reduced (22)Na(+) uptake and efflux by the CP. Steady-state extractions of a low and medium size molecular weight extracellular space marker, (14)C-mannitol and (3)H-polyethylene glycol, respectively, were significantly higher in CPs from old compared to young animals; however, there was no significant difference in steady-state extraction of a high molecular weight marker, (125)I-bovine serum albumin. This indicates increased passive BCSFB permeability for small and medium sized molecules in old sheep. CP redox activity was significantly lower in the old animals as assessed by the MTT assay, however, there was no significant difference in ATP content and energy charge of the CP with age suggesting adequate baseline energy reserve capacity. These data indicate that normal aging processes alter protein content in the CSF, CSF secretion, integrity of the BCSFB and Na(+) flux in the epithelial layer, which could impact on CSF homeostasis and turnover.

Dose-dependent transthyretin inhibition of T4 uptake from cerebrospinal fluid in sheep.

Transthyretin (TTR), synthesized by the choroid plexuses (CP) has an important role in transporting thyroxine from blood to cerebrospinal fluid (CSF). However, the role of TTR on thyroxine transport from CSF to either blood or brain is not clear. By using the incubated isolated ovine brain tissues technique, we found the CP accumulated most 125I-T4 compared to ventricular ependymal, frontal cortex or cerebellum. The accumulation was higher in the young CP than the old. There was dose-dependent inhibition by TTR on 125I-T4 accumulation in the brain tissues, and kinetics of T4 accumulation in presence of TTR was obtained by plotting a double reciprocal of B (bound) versus TTR concentration curve. The KD of 125I-T4 binding to TTR was higher in the CP compared to other tissues, suggesting that CP competes with TTR for T4 binding to a greater extent than the other tissues. Using the isolated perfused CP preparation, TTR significantly inhibited 125I-T4 efflux across CP from the CSF to blood side. Bovine serum albumin (BSA) was also able to inhibit 125I-T4 accumulation in the incubated tissues, but required higher concentrations to reach the level of inhibition seen with TTR. In conclusion, this study found a significant role for CSF TTR in preventing T4 loss to blood across the CP, and TTR inhibited both CP and selected brain tissue uptake in a dose-dependent manner. The physiological relevance of TTR may relate to preventing T4 loss from CSF and encouraging redistribution of hormone around the brain in CSF.

Leptin transport at the blood--cerebrospinal fluid barrier using the perfused sheep choroid plexus model.

Leptin is secreted by adipose tissue and thought to regulate appetite at the central level. Several studies have explored the central nervous system (CNS) entry of this peptide across the blood-brain and blood-cerebrospinal fluid (CSF) barriers in parallel, but this is the first to explore the transport kinetics of leptin across the choroid plexus (blood-CSF barrier) in isolation from the blood-brain barrier (BBB). This is important as the presence of both barriers can lead to ambiguous results from transport studies. The model used was the isolated Ringer perfused sheep choroid plexus. The steady-state extraction of [(125)I]leptin (7.5 pmol l(-1)) at the blood face of the choroid plexus was 21.1+/-5.7%, which was greater than extraction of the extracellular marker, giving a net cellular uptake for [(125)I]leptin (14.0+/-3.7%). In addition, trichloroacetic acid precipitable [(125)I] was detected in newly formed CSF, indicating intact protein transfer across the blood-CSF barrier. Human plasma concentrations of leptin are reported to be 0.5 nM. Experiments using 0.5 nM leptin in the Ringer produced a concentration of leptin in the CSF of 12 pM (similar to that measured in humans). [(125)I]Leptin uptake at the blood-plexus interface using the single-circulation paired tracer dilution technique (uptake in <60 s) indicated the presence of a saturable transport system, which followed Michaelis-Menten-type kinetics (K(m)=16.3+/-1.8 nM, V(max)=41.2+/-1.4 pmol min(-1) g(-1)), and a non-saturable component (K(d)=0.065+/-0.002 ml min(-1) g(-1)). In addition, secretion of new CSF by the choroid plexuses was significantly decreased with leptin present. This study indicates that leptin transport at the blood-CSF barrier is via saturable and non-saturable mechanisms and that the choroid plexus is involved in the regulation of leptin availability to the brain.

Ageing choroid plexus-cerebrospinal fluid system.

The impact of ageing on the choroid plexus (CP)-CSF circulatory system has largely been un-investigated, or has been of interest only in relation to neurological disease. This paper reviews the evidence for age-related changes to the CP-CSF system and compares changes with disease states where appropriate. The changes discussed include reduced ion transport capabilities, evidence for oxidative stress, altered hormone interactions, decreased CSF secretion rates in animal models and the contradictory nature of human data, reduced clearance of protein from CSF, and slower fluid turnover. The potential impacts of these changes are highlighted, including the possibility of reduced resistance to stress insults and slow clearance of toxic compounds from CSF with specific reference to amyloid peptide. Other impacts may include the reduced ability of CSF to act as a circulating medium for hormone and growth factors to reach their brain targets, and reduced homeostasis of CSF nutrients (amino acids, vitamins), which might influence brain interstitial fluid homeostasis.

A quantitative evaluation of the permeability of the blood brain barrier of portacaval shunted rats.

The integrity of the blood-brain barrier (BBB) was measured in male Sprague Dawley rats subjected to 16 weeks of portacaval shunting (PCS), the optimal time required for the cerebral changes to develop, by using an in situ brain perfusion technique. The penetration of a vascular space marker 14C mannitol, and labelled amino acids 3H-phenylalanine or 3H-glutamate were measured in brain and cerebrospinal fluid (CSF) using an in situ brain perfusion technique, over 2 or 20 minutes. The patency of the surgical shunt was confirmed by measurement of significantly increased plasma ammonia (131.5 +/- 14.8 micromol x l(-1)) and AST (159.5 +/- 19.9 IU x l(-1)) concentrations compared to controls 39.9 +/- 3.7*, and 82.5 +/- 6.6* respectively. Brain and CSF 14C-mannitol space (ml x 100g(-1)), was not increased by PCS where brain space was 1.31 +/- 0.27 mL x 100g(-1) compared to control 1.19 +/- 0.49 mL x 100g(-1), and CSF was 0.14 +/- 0.06 mL x 100g(-1) compared to control 0.15 +/- 0.05 (PCS n=10, control n=8). The uptake for 3H-glutamate, which is required for cerebral ammonia detoxification, was also unchanged in both brain and CSF. However, brain uptake of 3H-phenylalanine was significantly reduced from 871 +/- 80 microL x min(-1) x g(-1) to 356 +/- 154* microl x min(-1) x g(-1) (n=4), although there was no change in CSF uptake. These data suggest that there is no generalized breakdown of the blood-brain or blood-CSF barriers during PCS as assessed by mannitol penetration. The reduction in phenylalanine uptake into the brain may help stabilize high cerebral aromatic amino acid levels. *P<0.05, Two-tailed, Student's unpaired t-test.

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.

AVP V1 receptor-mediated decrease in Cl- efflux and increase in dark cell number in choroid plexus epithelium.

The cerebrospinal fluid (CSF)-generating choroid plexus (CP) has many V1 binding sites for arginine vasopressin (AVP). AVP decreases CSF formation rate and choroidal blood flow, but little is known about how AVP alters ion transport across the blood-CSF barrier. Adult rat lateral ventricle CP was loaded with 36Cl-, exposed to AVP for 20 min, and then placed in isotope-free artificial CSF to measure release of 36Cl-. Effect of AVP at 10(-12) to 10(-7) M on the Cl- efflux rate coefficient (in s-1) was quantified. Maximal inhibition (by 20%) of Cl- extrusion at 10(-9) M AVP was prevented by the V1 receptor antagonist [beta-mercapto-beta, beta-cyclopentamethyleneproprionyl1,O-Me-Tyr2,Arg8]vasopressin. AVP also increased by more than twofold the number of dark and possibly dehydrated but otherwise morphologically normal choroid epithelial cells in adult CP. The V1 receptor antagonist prevented this AVP-induced increment in dark cell frequency. In infant rats (1 wk) with incomplete CSF secretory ability, 10(-9) M AVP altered neither Cl- efflux nor dark cell frequency. The ability of AVP to elicit functional and structural changes in adult, but not infant, CP epithelium is discussed in regard to ion transport, CSF secretion, intracranial pressure, and hydrocephalus.

Toxic effects of beta-amyloid(25-35) on immortalised rat brain endothelial cell: protection by carnosine, homocarnosine and beta-alanine.

The effect of a truncated form of the neurotoxin beta-amyloid peptide (A beta25-35) on rat brain vascular endothelial cells (RBE4 cells) was studied in cell culture. Toxic effects of the peptide were seen at 200 microg/ml A beta using a mitochondrial dehydrogenase activity (MTT) reduction assay, lactate dehydrogenase release and glucose consumption. Cell damage could be prevented completely at 200 microg/ml A beta and partially at 300 microg/ml A beta, by the dipeptide carnosine. Carnosine is a naturally occurring dipeptide found at high levels in brain tissue and innervated muscle of mammals including humans. Agents which share properties similar to carnosine, such as beta-alanine, homocarnosine, the anti-glycating agent aminoguanidine, and the antioxidant superoxide dismutase (SOD), also partially rescued cells, although not as effectively as carnosine. We postulate that the mechanism of carnosine protection lies in its anti-glycating and antioxidant activities, both of which are implicated in neuronal and endothelial cell damage during Alzheimer's disease. Carnosine may therefore be a useful therapeutic agent.

Pluripotent protective effects of carnosine, a naturally occurring dipeptide.

Carnosine is a naturally occurring dipeptide (beta-alanyl-L-histidine) found in brain, innervated tissues, and the lens at concentrations up to 20 mM in humans. In 1994 it was shown that carnosine could delay senescence of cultured human fibroblasts. Evidence will be presented to suggest that carnosine, in addition to antioxidant and oxygen free-radical scavenging activities, also reacts with deleterious aldehydes to protect susceptible macromolecules. Our studies show that, in vitro, carnosine inhibits nonenzymic glycosylation and cross-linking of proteins induced by reactive aldehydes (aldose and ketose sugars, certain triose glycolytic intermediates and malondialdehyde (MDA), a lipid peroxidation product). Additionally we show that carnosine inhibits formation of MDA-induced protein-associated advanced glycosylation end products (AGEs) and formation of DNA-protein cross-links induced by acetaldehyde and formaldehyde. At the cellular level 20 mM carnosine protected cultured human fibroblasts and lymphocytes, CHO cells, and cultured rat brain endothelial cells against the toxic effects of formaldehyde, acetaldehyde and MDA, and AGEs formed by a lysine/deoxyribose mixture. Interestingly, carnosine protected cultured rat brain endothelial cells against amyloid peptide toxicity. We propose that carnosine (which is remarkably nontoxic) or related structures should be explored for possible intervention in pathologies that involve deleterious aldehydes, for example, secondary diabetic complications, inflammatory phenomena, alcoholic liver disease, and possibly Alzheimer's disease.

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.

Protective effects of carnosine against malondialdehyde-induced toxicity towards cultured rat brain endothelial cells.

Malondialdehyde (MDA) is a deleterious end-product of lipid peroxidation. The naturally-occurring dipeptide carnosine (beta-alanyl-L-histidine) is found in brain and innervated tissues at concentrations up to 20 mM. Recent studies have shown that carnosine can protect proteins against cross-linking mediated by aldehyde-containing sugars and glycolytic intermediates. Here we have investigated whether carnosine is protective against malondialdehyde-induced protein damage and cellular toxicity. The results show that carnosine can (1) protect cultured rat brain endothelial cells against MDA-induced toxicity and (2) inhibit MDA-induced protein modification (formation of cross-links and carbonyl groups).

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.

Potassium efflux from infant and adult rat choroid plexuses: effects of CSF anion substitution, N-ethylmaleimide and Cl transport inhibitors.

To analyze interdependent transport of K and Cl, we investigated Rb (K) efflux from in vitro choroid plexus (CP) in isotonic artificial CSF (aCSF) medium containing anions or agents that alter KCl transport. Lateral ventricle CP was loaded with 86Rb for release to enable calculation of the efflux rate coefficient, k. With Cl as the main anion in control aCSF, the k value for 86Rb (K) in CP of 1 week infant rats (0.177 min-1) was 19% lower than in adults (0.218 min-1) (P < 0.005). Replacing CSF Cl with NO3 or SCN, respectively, reduced k for K in infant CP by 73% and 43%; similar anion selectivity was observed in adult tissues (P < 0.05). N-Ethylmaleimide (NEM), which stimulates KCI cotransport, significantly enhanced K efflux in infants and adults. In adult CP, the KCl cotransport inhibitor, furosemide (1 mM), decreased K efflux by 23% or 65%, respectively, when aCSF had Cl or NO3 as the main anion. In infant rat CP, 0.1 mM bumetanide (another KCl cotransport inhibitor), reduced k for K by 65%, whereas the Cl channel blocker diphenylamine carboxylate (1 mM) did not significantly alter K efflux. The collective findings for rat CP indicate a substantial component of K efflux that is associated with Cl concentration and the Cl transport protein sensitive to loop diuretics and NEM. The Cl-dependent K efflux is present in infants.

Development of chloride transport by the rat choroid plexus, in vitro.

The uptake and efflux of 36Cl in the lateral ventricle choroid plexus of 1-7-week-old rats were measured to determine if Cl transport changed with age and if such transport responded to inhibitors of CSF secretion and ion transport. The steady-state (30 min) Cl uptakes were 148 +/- 9.4 nmol.mg-1 dry weight at 1 week and 139 +/- 7.0 nmol.mg-1 dry weight at 7 weeks, (P > 0.05). The 36Cl efflux was significantly slower in 1 and 2 week plexuses compared to more mature tissues (P < 0.01) with k (rate coefficient) = 0.029 +/- 0.004 s-1, t1/2 = 24.1 +/- 3.3 s at 1 week and k = 0.041 +/- 0.003 s-1, t1/2 = 17.4 +/- 1.3 s at 7 weeks. 36Cl efflux at 1 week was unaffected by acetazolamide, bumetanide and DIDS (4,4 diisothiocyanato-stilbene-2,2 disulphonic acid), however, all these drugs substantially reduced efflux from 2, 3 and 7 week choroid plexuses. In contrast, the Cl conductance blocker, DPC (diphenylamine carboxylate) at 10(-4) M reduced 36Cl efflux from both 1 and 7 week tissues by 43% and 39%, respectively. These findings suggest that some transport systems responsible for movement of Cl out of the epithelium are either absent or less functional in the immature rat choroid plexus and may account for the relatively low level of CSF secretion in younger animals. The unidirectional efflux of 36Cl, J, was calculated for 1 week and adult rats, as a function of the choroid plexus volume to surface area ratio (V/A).(ABSTRACT TRUNCATED AT 250 WORDS)

Saturable uptake of [125I]L-triiodothyronine at the basolateral (blood) and apical (cerebrospinal fluid) sides of the isolated perfused sheep choroid plexus.

The uptake of 125I-labelled L-triiodothyronine (T3) was measured on the blood side of the isolated perfused choroid plexus of the sheep using steady-state and single-circulation paired tracer techniques. The steady-state uptake of T3 was 33.5% (perfusion fluid protein content was 0.05 g.dl-1) which could be reduced to 9.4% in the presence of 500 microM unlabelled T3 showing partial saturation. The CSF to blood steady-state [125I]T3 measurements gave plasma/CSF ratio, R%, of 24.6 +/- 4.8% which was reduced to 9.8 +/- 2.1% in the presence of 500 microM unlabelled T3 in the mock CSF. The transport of T3 across the blood face of the choroid plexus and the CSF to blood transport, failed to show sodium dependence. Using the single circulation paired tracer technique, the initial uptake in less than 60 s, Umax of [125I]T3 was 50.4 +/- 3.9% relative to the extracellular marker [3H]D-mannitol. However, when 250 microM unlabelled T3 was present, Umax was reduced by 66%, although further significant inhibition at higher concentrations was not observed. Uptake of T3 at the blood side of the choroid plexus was partially saturated in the presence of unlabelled reverse T3 and DT3, suggesting little uptake stereospecificity. Unlabelled thyroxine (T4) and the amino acid analogues cycloleucine (aminocyclopentane-1-carboxylic acid) and BCH (beta-2-aminobicyclo-[2,2.1]-heptane-2-carboxylic acid) each reduced [125I]T3 uptake significantly, but not to the same degree as T3 stereoisomers. The neutral amino acids alanine and phenylalanine, had no effect on uptake.(ABSTRACT TRUNCATED AT 250 WORDS)