Biomedical subjects

Malcolm B Segal

Publications and source records attributed to Malcolm B Segal.

9 recordsLinked to original sources

PubMed2006

Role of transthyretin in thyroxine transfer from cerebrospinal fluid to brain and choroid plexus.

The transport of 125I-labeled thyroxine (T4) from the cerebrospinal fluid (CSF) into brain and choroid plexus (CP) was measured in anesthetized rabbit [0.5 mg/kg medetomidine (Domitor) and 10 mg/kg pentobarbitonal sodium (Sagatal) iv] using the ventriculocisternal (V-C) perfusion technique. 125I-labeled T4 contained in artificial CSF was continually perfused into the lateral ventricles for up to 4 h and recovered from the cisterna magna. The %recovery of 125I-labeled T4 from the aCSF was 47.2+/-5.6% (n=10), indicating removal of 125I-labeled T4 from the CSF. The recovery increased to 53.2+/-6.3% (n=4) and 57.8+/-14.8% (n=3), in the presence of 100 and 200 microM unlabeled-T4, respectively (P<0.05), indicating a saturable component to T4 removal from CSF. There was a large accumulation of 125I-labeled T4 in the CP, and this was reduced by 80% in the presence of 200 microM unlabeled T4, showing saturation. In the presence of the thyroid-binding protein transthyretin (TTR), more 125I-labeled T4 was recovered from CSF, indicating that the binding protein acted to retain T4 in CSF. However, 125I-labeled T4 uptake into the ependymal region (ER) of the frontal cortex also increased by 13 times compared with control conditions. Elevation was also seen in the hippocampus (HC) and brain stem. Uptake was significantly inhibited by the presence of endocytosis inhibitors nocodazole and monensin by >50%. These data suggest that the distribution of T4 from CSF into brain and CP is carrier mediated, TTR dependent, and via RME. These results support a role for TTR in the distribution of T4 from CSF into brain sites around the ventricular system, indicating those areas involved in neurogenesis (ER and HC).

Animals↗

PubMed2006

Uneven distribution of nucleoside transporters and intracellular enzymatic degradation prevent transport of intact [14C] adenosine across the sheep choroid plexus epithelium as a monolayer in primary culture.

BACKGROUND: Efflux transport of adenosine across the choroid plexus (CP) epithelium might contribute to the homeostasis of this neuromodulator in the extracellular fluids of the brain. The aim of this study was to explore adenosine transport across sheep CP epithelial cell monolayers in primary culture. METHODS: To explore transport of adenosine across the CP epithelium, we have developed a method for primary culture of the sheep choroid plexus epithelial cells (CPEC) on plastic permeable supports and analysed [14C] adenosine transport across this cellular layer, [14C] adenosine metabolism inside the cells, and cellular uptake of [14C] adenosine from either of the chambers. The primary cell culture consisted of an enriched epithelial cell fraction from the sheep fourth ventricle CP and was grown on laminin-precoated filter inserts. RESULTS AND CONCLUSION: CPEC grew as monolayers forming typical polygonal islands, reaching optical confluence on the third day after the seeding. Transepithelial electrical resistance increased over the time after seeding up to 85 +/- 9 Omega cm2 at day 8, while permeability towards [14C] sucrose, a marker of paracellular diffusion, simultaneously decreased. These cells expressed some features typical of the CPEC in situ, including three nucleoside transporters at the transcript level that normally mediate adenosine transport across cellular membranes. The estimated permeability of these monolayers towards [14C] adenosine was low and the same order of magnitude as for the markers of paracellular diffusion.However, inhibition of the intracellular enzymes, adenosine kinase and adenosine deaminase, led to a significant increase in transcellular permeability, indicating that intracellular phosphorylation into nucleotides might be a reason for the low transcellular permeability. HPLC analysis with simultaneous detection of radioactivity revealed that [14C] radioactivity which appeared in the acceptor chamber after the incubation of CPEC monolayers with [14C] adenosine in the donor chamber was mostly present as [14C] hypoxanthine, a product of adenosine metabolic degradation. Therefore, it appears that CPEC in primary cultures act as an enzymatic barrier towards adenosine. Cellular uptake studies revealed that concentrative uptake of [14C] adenosine was confined only to the side of these cells facing the upper or apical chamber, indicating uneven distribution of nucleoside transporters.

Journal Article↗

PubMed2005

Hyperammonaemia reduces intracellular 22Na (sodium) ion and extracellular 86Rb ion concentrations in the blood-brain barrier of the rat.

Ammonia may be the major cerebral intoxicant responsible for the increased general or passive permeability of the blood-brain barrier (BBB) leading to the cerebral edema associated with acute liver failure. The present study investigated the effects of ammonia, as NH4+, on Na+ (22Na), K+ (86Rb), and 14C-mannitol uptake in the BBB. An in situ isolated perfused rat brain preparation was used to study the action of 1 mM ammonium acetate in Krebs'-Ringer perfusate. Passive water transport in the brain was studied by 14C-labeled mannitol uptake, a usually nondiffusible marker and active water transport by 22Na and 86Rb uptake. NH4+ significantly reduced 14C-mannitol uptake into the choroid plexus (P < 0.001) and increased it in the CSF (P < 0.05). Decreased 86Rb was measured in whole brain (P < 0.01) and CSF. However, no effect was observed in brain parenchyma, endothelium or choroid plexus thereby suggesting an increased efflux of 86Rb to the interstitial fluid. NH+ increased Na+ uptake into all areas of the brain studied. NH4+ does not increase the passive permeability into the BBB and was decreased in the choroid plexus. The increased 22Na+ uptake was substantiated by the observed decreases in 86Rb uptake in whole brain and CSF. This suggested NH4+ stimulates the Na+/K+ pump and increases extracellular Na+ concentrations and possibly intracellular concentrations with a concomitant decrease in K+ concentrations. These observations may provide a basis for the explanation of NH+ toxicity during hepatic encephalopathy and liver failure-induced cerebral edema.

Ammonia↗

PubMed2005

Decrease of transthyretin synthesis at the blood-cerebrospinal fluid barrier of old sheep.

Transthyretin (TTR), synthesized by the choroid plexus (CP) and secreted into cerebrospinal fluid (CSF), is involved in thyroxine (T4) transport and chelation of beta-amyloid peptide, attenuating neurotoxicity. To characterize age-related changes in TTR synthesis, CSF and CPs were collected from young adult (1-2 years) and old (>8 years) sheep anesthetized with thiopentone sodium. TTR in old sheep CSF was low compared to young (n = 4 each); however, CP messenger RNA (mRNA) for TTR did not change. CPs were perfused with Ringer containing 14C-leucine to assess de novo protein synthesis, or with 125I-T4 to assess T4 transport. Protein synthesis, including TTR, was reduced in old sheep CP and in newly secreted CSF. 125I-T4 Vmax and Kd (but not Km) were reduced in old sheep CP. These age-related changes suggest reduced capacity of CP to maintain CSF T4 homeostasis and could also reduce chelation of beta-amyloid and be an added risk for Alzheimer's disease.

Aging↗

PubMed2004

The structure of the choroid plexus and the physiology of the choroid plexus epithelium.

The choroid plexuses (CPs) are leaf-like highly vascular structures laying in the ventricles. The main function of choroid plexuses is the production of the cerebrospinal fluid (CSF). Although CPs have a unique distribution of ion transporters/channels, the mechanism of CSF production is similar to the production of fluids in other epithelia and is based on energy released from ATP hydrolysis, which drives unidirectional flux of ions accompanied by movement of water by osmosis. The CPs have an important role in the homeostasis of nutrients in the CSF since the kinetic parameters of glucose and amino acid (AA) transport across the CPs are the main reason for the low concentration of these molecules in the CSF. The CPs appear to be source of CSF-borne hormones and growth factors, including insulin-like growth factor II (IGF II), vasopressin (VP) and transforming growth factor beta1 (TGF-beta1). The CPs also synthesise the thyroid transporting protein transthyretin and transferrin and can chelate heavy metals.

Amino Acids↗

PubMed2003

Transport of L-[125I]thyroxine by in situ perfused ovine choroid plexus: inhibition by lead exposure.

Lead (Pb) exposure hinders brain development in children by mechanisms that remain unknown. Previous evidence shows that sequestration of Pb in the choroid plexus lowers the production and secretion of transthyretin (TTR), a thyroxine (T4) transport protein, from the choroid plexus into the cerebrospinal fluid (CSF). This study was undertaken to characterize the uptake kinetics of T4 by the choroid plexus and to determine if in vivo Pb exposure altered the T4 uptake in an in situ perfused ovine choroid plexus model. Sheep received i.p. injections of Pb acetate (20 mg Pb/kg) or Na acetate (as the controls) every 48 h for a period of 16 d. The [125I]T4 uptake was determined by a paired-tracer perfusion method using 0.5 microCi [125I]T4 and 2 microCi [14C]mannitol at various concentrations of unlabeled T4 (trace to 20 microM). The flux of [125I]T4 into the choroid plexus followed Michaelis-Menten kinetics with the maximum flux (Vmax) of 56.6 nmol/min/g and half-saturation constant (Km) of 10.7 mumol/L, suggesting an evident saturable influx of T4 into the choroid epithelium. In vivo Pb exposure in these sheep resulted in a significant accumulation of Pb in the choroid plexus and hippocampus. Pb treatment diminished the Vmax by 63.7% of control, but did not alter Km. The maximal cellular uptake (Umax) and net uptake (Unet) in Pb-treated animals were 2.1-fold and 1.9-fold, respectively, lower than those of control. Exposure to Pb, however, did not significantly change the flow rate through the choroid plexus. Data suggest that the choroid plexus may serve as a significant site for T4 transport into the CSF, and Pb exposure may hinder the influx of T4 from the blood into the choroid plexus.

Animals↗

PubMed2002

The kinetics of hypoxanthine transport across the perfused choroid plexus of the sheep.

The uptake of principal salvageable nucleobase hypoxanthine was investigated across the basolateral membrane of the sheep choroid plexus (CP) perfused in situ. The results suggest that hypoxanthine uptake was Na+-independent, which means that transport system on the basolateral membrane can mediate the transport in both directions. Although the unlabelled nucleosides adenosine and inosine markedly reduce the transport it seems that this inhibition was due to nucleoside degradation into nucleobases in the cells, since non-metabolised nucleoside analogue NBTI did not inhibit the transport. The presence of adenine also inhibits hypoxanthine uptake while the addition of the pyrimidines does not show any effect, so it seems that the transport of purine nucleobases through basolateral membrane is mediated via a common transporter which is different from the nucleoside transporters. The inclusion of allopurinol in the perfusion fluid did not change the value and general shape of the curve for the uptake which suggest that degradation of hypoxanthine into xanthine and uric acid does not occur in the CP. The capacity of the CP basolateral membrane to transport hypoxanthine is high (90.63+/-3.79 nM/min/g) and close to the values obtained for some essential amino acids by the CP and blood-brain barrier, while the free diffusion is negligible. The derived value of Km (20.72+/-2.42 microM) is higher than the concentration of hypoxanthine in the sheep plasma (15.61+/-2.28 microM) but less than a half of the concentration in the CSF, which indicates that the transport system at basolateral membrane mostly mediates the efflux of hypoxanthine from the cerebrospinal fluid in vivo.

Adenine↗

PubMed2002

The efflux of purine nucleobases and nucleosides from the rat brain.

The efflux of purine nucleobases and their nucleosides from the rat brain was investigated using the brain efflux index (BEI) method. Calculated BEI values showed that purine nucleobases had very rapid initial efflux after the intracerebral injection, which was followed by the slower efflux due to the intracellular trapping of labelled molecules and confirmed by the capillary depletion technique. The efflux of ribonucleosides was much slower than the efflux of nucleobases and the structure of the sugar moiety seemed to be important, since a significant difference in the efflux velocity between ribo- and deoxyribonucleosides was observed. The results of self- and cross-inhibition studies suggested that the efflux of test molecules was saturable and that purines shared the same transport system on the abluminal side of the blood-brain barrier.

Adenosine↗

PubMed2002

The linkage of glucose to tiazofurin decreases in vitro uptake into rat glioma C6 cells.

The aim of this study was to analyse the uptake of the synthetic nucleoside tiazofurin and glucoso-linker-tiazofurin conjugate (GLTC) into rat C6 glioma cells in vitro. Results indicated that C6 cells accumulated [3H] tiazofurin slowly with time and that accumulation was reduced by the presence of unlabelled GLTC in the medium which implies that GLTC competes with tiazofurin for transport sites. Uptake of [14C] 2 deoxy-glucose into these cells was very rapid and was not affected by the presence of unlabelled GLTC. To prove the true rate of uptake, the HPLC analysis of cellular extract was performed. After the 360 min of incubation in medium that contained 0.15 mM of tiazofurin, the sum of the concentration of tiazofurin and it's metabolite thiazole-adenine dinucleotide (TAD) in the cells was a total of approximately 4.8% of the amount added to each flask. After the same period of incubation in medium which contained 0.15 mM of GLTC, the sum of concentrations of conjugate, free tiazofurin and TAD represented less than 1/3 of the total concentration measured after the incubation with free tiazofurin and was further reduced in the presence of dipyridamole. Therefore, it can be concluded that GLTC shows some affinity for the nucleoside transporter, but the actual rate of uptake is low.

Animals↗