Expression and possible role of creatine transporter in the brain and at the blood-cerebrospinal fluid barrier as a transporting protein of guanidinoacetate, an endogenous convulsant

Little is known about the cerebral distribution and clearance of guanidinoacetate (GAA), the accumulation of which induces convulsions. The purpose of the present study was to identify creatine transporter (CRT)-mediated GAA transport and to clarify its cerebral expression and role in GAA efflux transport at the blood-cerebrospinal fluid barrier (BCSFB). CRT mediated GAA transport with a K(m) value of 269 microM/412 microM which was approximately 10-fold greater than that of CRT for creatine. There was wide and distinct cerebral expression of CRT and localization of CRT on the brush-border membrane of choroid plexus epithelial cells. The in vivo elimination clearance of GAA from the CSF was 13-fold greater than that of d-mannitol reflecting bulk flow of the CSF. This process was partially inhibited by creatine. The characteristics of GAA uptake by isolated choroid plexus and an immortalized rat choroid plexus epithelial cell line (TR-CSFB cells) used as an in vitro model of BCSFB are partially consistent with those of CRT. These results suggest that CRT plays a role in the cerebral distribution of GAA and GAA uptake by the choroid plexus. However, in the presence of endogenous creatine in the CSF, CRT may make only a limited contribution to the GAA efflux transport at the BCSFB.     

Aminothiazoles inhibit RANKL‐ and LPS‐mediated osteoclastogenesis and PGE2 production in RAW 264.7 cells

Periodontitis is characterized by chronic inflammation and osteoclast‐mediated bone loss regulated by the receptor activator of nuclear factor‐κB (RANK), RANK ligand (RANKL) and osteoprotegerin (OPG). The aim of this study was to investigate the effect of aminothiazoles targeting prostaglandin E synthase‐1 (mPGES‐1) on RANKL‐ and lipopolysaccharide (LPS)‐mediated osteoclastogenesis and prostaglandin E₂ (PGE₂) production in vitro using the osteoclast precursor RAW 264.7 cells. RAW 264.7 cells were treated with RANKL or LPS alone or in combination with the aminothiazoles 4‐([4‐(2‐naphthyl)‐1,3‐thiazol‐2‐yl]amino)phenol (TH‐848) or 4‐(3‐fluoro‐4‐methoxyphenyl)‐N‐(4‐phenoxyphenyl)‐1,3‐thiazol‐2‐amine (TH‐644). Aminothiazoles significantly decreased the number of multinucleated tartrate‐resistant acid phosphatase (TRAP)‐positive osteoclast‐like cells in cultures of RANKL‐ and LPS‐stimulated RAW 264.7 cells, as well as reduced the production of PGE₂ in culture supernatants. LPS‐treatment induced mPGES‐1 mRNA expression at 16 hrs and the subsequent PGE₂ production at 72 hrs. Conversely, RANKL did not affect PGE₂ secretion but markedly reduced mPGES‐1 at mRNA level. Furthermore, mRNA expression of TRAP and cathepsin K (CTSK) was reduced by aminothiazoles in RAW 264.7 cells activated by LPS, whereas RANK, OPG or tumour necrosis factor α mRNA expression was not significantly affected. In RANKL‐activated RAW 264.7 cells, TH‐848 and TH‐644 down‐regulated CTSK but not TRAP mRNA expression. Moreover, the inhibitory effect of aminothiazoles on PGE₂ production was also confirmed in LPS‐stimulated human peripheral blood mononuclear cell cultures. In conclusion, the aminothiazoles reduced both LPS‐ and RANKL‐mediated osteoclastogenesis and PGE₂ production in RAW 264.7 cells, suggesting these compounds as potential inhibitors for treatment of chronic inflammatory bone resorption, such as periodontitis.     

Aminothiazoles inhibit osteoclastogenesis and PGE2 production in LPS‐stimulated co‐cultures of periodontal ligament and RAW 264.7 cells,and RANKL‐mediated osteoclastogenesis and bone resorption in PBMCs

Inflammatory mediator prostaglandin E₂ (PGE₂) contributes to bone resorption in several inflammatory conditions including periodontitis. The terminal enzyme, microsomal prostaglandin E synthase‐1 (mPGES‐1) regulating PGE₂ synthesis is a promising therapeutic target to reduce inflammatory bone loss. The aim of this study was to investigate effects of mPGES‐1 inhibitors, aminothiazoles TH‐848 and TH‐644, on PGE₂ production and osteoclastogenesis in co‐cultures of periodontal ligament (PDL) and osteoclast progenitor cells RAW 264.7, stimulated by lipopolysaccharide (LPS), and bone resorption in RANKL‐mediated peripheral blood mononuclear cells (PBMCs). PDL and RAW 264.7 cells were cultured separately or co‐cultured and treated with LPS alone or in combination with aminothiazoles. Multinucleated cells stained positively for tartrate‐resistant acid phosphatase (TRAP) were scored as osteoclast‐like cells. Levels of PGE₂, osteoprotegerin (OPG) and interleukin‐6, as well as mRNA expression of mPGES‐1, OPG and RANKL were analysed in PDL cells. PBMCs were treated with RANKL alone or in combination with aminothiazoles. TRAP‐positive multinucleated cells were analysed and bone resorption was measured by the CTX‐I assay. Aminothiazoles reduced LPS‐stimulated osteoclast‐like cell formation both in co‐cultures and in RAW 264.7 cells. Additionally, aminothiazoles inhibited PGE₂ production in LPS‐stimulated cultures, but did not affect LPS‐induced mPGES‐1, OPG or RANKL mRNA expression in PDL cells. In PBMCs, inhibitors decreased both osteoclast differentiation and bone resorption. In conclusion, aminothiazoles reduced the formation of osteoclast‐like cells and decreased the production of PGE₂ in co‐cultures as well as single‐cell cultures. Furthermore, these compounds inhibited RANKL‐induced bone resorption and differentiation of PBMCs, suggesting these inhibitors for future treatment of inflammatory bone loss such as periodontitis.     

Transport systems of serine at the brain barriers and in brain parenchymal cells

D-Serine is a co-agonist for NMDA-type glutamate receptors. Although D-serine levels in CSF and interstitial fluid (ISF) affect CNS function, the regulatory system remains to be fully understood. Therefore, the purpose of this study was to investigate d-serine transport across the blood-brain barrier (BBB) and blood-CSF barrier (BCSFB) and in brain parenchymal cells. D-Serine microinjected into the cerebrum was not eliminated, suggesting a negligible contribution of D-serine efflux transport at the BBB. In contrast, D-serine was taken up from the circulating blood across the BBB via a carrier-mediated process. D-Serine elimination clearance from CSF was fourfold greater than that of d-mannitol, which is considered to reflect CSF bulk flow. The characteristics of D-serine uptake by isolated choroid plexus were consistent with those of Na(+)-independent alanine-serine-cysteine transporter 1 (asc-1). Uptake of D-serine by brain slices appeared to occur predominantly via asc-1 and Na(+)-dependent alanine-serine-cysteine transporter 2. These findings suggest that the regulatory system of D-serine levels in ISF and CSF involves (i) asc-1 at the BCSFB, acting as a major pathway of D-serine elimination from the CSF, (ii) blood-to-brain and blood-to-CSF influx transport of D-serine across the BBB and BCSFB, and (iii) concentrative uptake of D-serine by brain parenchymal cells.     

The movement of sulfate and thiosulfate into in vivo choroid plexus

The accumulation of sulfate (SO₄?) and thiosulfate (S₂O₃?) in the choroid plexus, brain, and cerebrospinal fluid (CSF) of the rat was measured at various plasma levels of these anions. Increasing the plasma SO₄ ? or S₂O₃ ? concentration levels 40- and 580-fold, respectively, resulted in a linear increase in CSF, brain and choroid plexus concentration of these ions. The relationship between the concentration of these ions in CSF and choroid plexus was also approximately linear over a wide CSF concentration range. In addition, S₂O₃? did not appear to influence the relation between the concentration of SO₄? in choroid plexus and CSF. The results seem to indicate that there is no saturation of the mechanism responsible for maintaining the low SO₄? or S₂O₃? concentration in CSF nor does there appear to be competition between these anions for clearance from the CSF. These findings are in conflict with data supporting the active transport of SO₄? and S₂O₃? from the CSF across the CSF-blood barrier (choroid plexus).     

Down-regulation of microsomal prostaglandin E2 synthase-1 in adipose tissue by high-fat feeding

Objective: Prostaglandin (PG)E₂ is a lipid mediator implicated in inflammatory diseases and in the regulation of lipolysis and adipocyte differentiation. This work was, thus, undertaken to study the regulation of the various PGE₂ synthases (PGESs) in obesity. Research Methods and Procedures: C57Bl/6 mice were subjected to a high‐fat or regular diet for 12 weeks. The levels of PGE₂ in white adipose tissue (WAT) of lean and obese mice were quantified by liquid chromatography‐mass spectrometry, and the change in expression of the three major PGES caused by diet‐induced obesity was characterized by Western blotting. Human preadipocytes and 3T3‐L1 cells were used to assess the expression of microsomal prostaglandin E₂ synthase‐1 (mPGES‐1) during adipogenesis. Results: mPGES‐1, mPGES‐2, and cytosolic PGES proteins were all detected in WAT of lean animals. mPGES‐1 was expressed at higher levels in WAT than in any other tissues examined and was more abundant (3‐ to 4‐fold) in epididymal (visceral) compared with inguinal (subcutaneous) WAT. Expression of mPGES‐1 was also detected in undifferentiated and differentiated 3T3‐L1 cells and in human primary subcutaneous preadipocytes at all stages of adipogenesis. The mPGES‐1 protein was substantially down‐regulated in epididymal and inguinal WAT of obese mice, whereas mPGES‐2 and cytosolic PGES remained relatively stable. Concordantly, the PGE₂ levels in obese inguinal WAT were significantly lower than those of lean animals. Discussion: These data suggest that mPGES‐1 is the major form of PGESs contributing to the synthesis of PGE₂ in WAT and that its down‐regulation might be involved in the alterations of lipolysis and adipogenesis associated with obesity.     

Glymphatic distribution of CSF-derived apoE into brain is isoform specific and suppressed during sleep deprivation

Background

Apolipoprotein E (apoE) is a major carrier of cholesterol and essential for synaptic plasticity. In brain, it’s expressed by many cells but highly expressed by the choroid plexus and the predominant apolipoprotein in cerebrospinal fluid (CSF). The role of apoE in the CSF is unclear. Recently, the glymphatic system was described as a clearance system whereby CSF and ISF (interstitial fluid) is exchanged via the peri-arterial space and convective flow of ISF clearance is mediated by aquaporin 4 (AQP4), a water channel. We reasoned that this system also serves to distribute essential molecules in CSF into brain. The aim was to establish whether apoE in CSF, secreted by the choroid plexus, is distributed into brain, and whether this distribution pattern was altered by sleep deprivation.

Methods

We used fluorescently labeled lipidated apoE isoforms, lenti-apoE3 delivered to the choroid plexus, immunohistochemistry to map apoE brain distribution, immunolabeled cells and proteins in brain, Western blot analysis and ELISA to determine apoE levels and radiolabeled molecules to quantify CSF inflow into brain and brain clearance in mice. Data were statistically analyzed using ANOVA or Student’s t- test.

Results

We show that the glymphatic fluid transporting system contributes to the delivery of choroid plexus/CSF-derived human apoE to neurons. CSF-delivered human apoE entered brain via the perivascular space of penetrating arteries and flows radially around arteries, but not veins, in an isoform specific manner (apoE2?>?apoE3?>?apoE4). Flow of apoE around arteries was facilitated by AQP4, a characteristic feature of the glymphatic system. ApoE3, delivered by lentivirus to the choroid plexus and ependymal layer but not to the parenchymal cells, was present in the CSF, penetrating arteries and neurons. The inflow of CSF, which contains apoE, into brain and its clearance from the interstitium were severely suppressed by sleep deprivation compared to the sleep state.

Conclusions

Thus, choroid plexus/CSF provides an additional source of apoE and the glymphatic fluid transporting system delivers it to brain via the periarterial space. By implication, failure in this essential physiological role of the glymphatic fluid flow and ISF clearance may also contribute to apoE isoform-specific disorders in the long term.

     

Altered clearance of beta-amyloid from the cerebrospinal fluid following subchronic lead exposure in rats: Roles of RAGE and LRP1 in the choroid plexus

Formation of amyloid plaques is the hallmark of Alzheimer’s disease. Our early studies show that lead (Pb) exposure in PDAPP transgenic mice increases β-amyloid (Aβ) levels in the cerebrospinal fluid (CSF) and hippocampus, leading to the formation of amyloid plaques in mouse brain. Aβ in the CSF is regulated by the blood-CSF barrier (BCB) in the choroid plexus. However, the questions as to whether and how Pb exposure affected the influx and efflux of Aβ in BCB remained unknown. This study was conducted to investigate whether Pb exposure altered the Aβ efflux in the choroid plexus from the CSF to blood, and how Pb may affect the expression and subcellular translocation of two major Aβ transporters, i.e., the receptor for advanced glycation end-products (RAGE) and the low density lipoprotein receptor protein-1 (LRP1) in the choroid plexus. Sprague-Dawley rats received daily oral gavage at doses of 0, 14 (low-dose), and 27 (high-dose) mg Pb/kg as Pb acetate, 5 d/wk, for 4 or 8 wks. At the end of Pb exposure, a solution containing Aβ₄₀ (2.5 μg/mL) was infused to rat brain via a cannulated internal carotid artery. Subchronic Pb exposure at both dose levels significantly increased Aβ levels in the CSF and choroid plexus (p < 0.05) by ELISA. Confocal data showed that 4-wk Pb exposures prompted subcellular translocation of RAGE from the choroidal cytoplasm toward apical microvilli. Furthermore, it increased the RAGE expression in the choroid plexus by 34.1 % and 25.1 % over the controls (p < 0.05) in the low- and high- dose groups, respectfully. Subchronic Pb exposure did not significantly affect the expression of LRP1; yet the high-dose group showed LRP1 concentrated along the basal lamina. The data from the ventriculo-cisternal perfusion revealed a significantly decreased efflux of Aβ₄₀ from the CSF to blood via the blood-CSF barrier. Incubation of freshly dissected plexus tissues with Pb in artificial CSF supported a Pb effect on increased RAGE expression. Taken together, these data suggest that Pb accumulation in the choroid plexus after subchronic exposure reduces the clearance of Aβ from the CSF to blood by the choroid plexus, which, in turn, leads to an increase of Aβ in the CSF. Interaction of Pb with RAGE and LRP1 in choroidal epithelial cells may contribute to the altered Aβ transport by the blood-CSF barrier in brain ventricles.     

Glutaric aciduria type I and methylmalonic aciduria: Simulation of cerebral import and export of accumulating neurotoxic dicarboxylic acids in in vitro models of the blood–brain barrier and the choroid plexus

Intracerebral accumulation of neurotoxic dicarboxylic acids (DCAs) plays an important pathophysiological role in glutaric aciduria type I and methylmalonic aciduria. Therefore, we investigated the transport characteristics of accumulating DCAs – glutaric (GA), 3-hydroxyglutaric (3-OH-GA) and methylmalonic acid (MMA) – across porcine brain capillary endothelial cells (pBCEC) and human choroid plexus epithelial cells (hCPEC) representing in vitro models of the blood–brain barrier (BBB) and the choroid plexus respectively. We identified expression of organic acid transporters 1 (OAT1) and 3 (OAT3) in pBCEC on mRNA and protein level. For DCAs tested, transport from the basolateral to the apical site (i.e. efflux) was higher than influx. Efflux transport of GA, 3-OH-GA, and MMA across pBCEC was Na⁺-dependent, ATP-independent, and was inhibited by the OAT substrates para-aminohippuric acid (PAH), estrone sulfate, and taurocholate, and the OAT inhibitor probenecid. Members of the ATP-binding cassette transporter family or the organic anion transporting polypeptide family, namely MRP2, P-gp, BCRP, and OATP1B3, did not mediate transport of GA, 3-OH-GA or MMA confirming the specificity of efflux transport via OATs. In hCPEC, cellular import of GA was dependent on Na⁺-gradient, inhibited by NaCN, and unaffected by probenecid suggesting a Na⁺-dependent DCA transporter. Specific transport of GA across hCPEC, however, was not found. In conclusion, our results indicate a low but specific efflux transport for GA, 3-OH-GA, and MMA across pBCEC, an in vitro model of the BBB, via OAT1 and OAT3 but not across hCPEC, an in vitro model of the choroid plexus.     

Riboflavin Homeostasis in the Central Nervous System

Abstract: The mechanisms by which riboflavin, which is not synthesized in mammals, enters and leaves brain, CSF, and choroid plexus were investigated by injecting [¹⁴C]riboflavin intravenously or intraventricularly. Tracer amounts of [¹⁴C]riboflavin with or without FMN were infused intravenously at a constant rate into normal, starved, or probenecid-pretreated rabbits. At 3 h, [¹⁴C]riboflavin readily entered choroid plexus and brain, and, to a much lesser extent, CSF. Over 85% of the [¹⁴C]riboflavin in brain and choroid plexus was present as [¹⁴C]FMN and [¹⁴C]FAD. The addition of 0.2 mmol/kg FMN to the infusate markedly depressed the relative entry of [¹⁴C]riboflavin into brain, choroid plexus, and, less so, CSF, whereas starvation increased the relative entry of [¹⁴C]riboflavin into brain and choroid plexus. After intraventricular injection (2 h), most of the [¹⁴C]riboflavin was extremely rapidly cleared from CSF into blood. Some of the [¹⁴C]riboflavin entered brain, where over 85% of the ¹⁴C was present as [¹⁴C]FMN plus [¹⁴C]FAD. The addition of 1.2³μmol FAD (which was rapidly hydrolyzed to riboflavin) to the injectate decreased the clearance of [¹⁴C]riboflavin from CSF and the phosphorylation of [¹⁴C]riboflavin in brain. Probenecid in the injectate also decreased the clearance of [¹⁴C]riboflavin from CSF. These results show that the control of entry and exit of riboflavin is the mechanism, at least in part, by which total riboflavin levels in brain cells and CSF are regulated. Penetration of riboflavin through the blood-brain barrier, saturable efflux of riboflavin from CSF, and saturable entry of riboflavin into brain cells are three distinct parts of the homeostatic system for total riboflavin in the central nervous system.     

Microsomal Prostaglandin E Synthase Type 2 (mPGES2) Is a Glutathione-dependent Heme Protein,and Dithiothreitol Dissociates the Bound Heme to Produce Active Prostaglandin E2 Synthase in Vitro

An x-ray study indicated that microsomal prostaglandin E synthase type 2 (mPGES2) is a heme-bound protein and catalyzes prostaglandin (PG) H₂ degradation, but not PGE₂ formation (Yamada, T., and Takusagawa, F. (2007) Biochemistry 46, 8414–8424). In response to the x-ray study, Watanabe et al. claimed that mPGES2 is a heme-free protein and that both the heme-free and heme-bound proteins have PGE₂ synthesis activity in the presence of dithiothreitol (Watanabe, K., Ito, S., and Yamamoto, S. (2008) Biochem. Biophys. Res. Commun. 367, 782–786). To resolve the contradictory results, the heme-binding scheme of mPGES2 was further characterized in vivo and in vitro by absorption and fluorescence spectroscopies. A substantial amount of heme-bound mPGES2 was detected in cell extracts. The heme content in mPGES2 was increased along with an increase in Fe³⁺ in the culture medium. Heme-free mPGES2 was converted to the heme-bound form by mixing it with pig liver extract, indicating that mPGES2 is capable of forming a complex with heme in mammalian cells. Heme binds to mPGES2 only in the presence of glutathione. The newly determined heme dissociation constant (2.9 nm) supports strongly that mPGES2 is a heme-bound protein in vivo. The bound heme was not dissociated by oxidation by H₂O₂ or reduction by glutathione or 2-mercaptoethanol. However, reduction by dithiothreitol (an artificial reducing compound) induced the bound heme to dissociate from mPGES2 and released heme-free mPGES2, which exhibited PGE₂ synthesis activity in vitro. Imidazole bound to mPGES2 by stacking on the bound heme and inhibited heme oxidation by H₂O₂ and reduction by dithiothreitol.     

Morphological Ground of Formation of the Brain Choroid Plexus Blood-Cerebrospinal Fluid Barrier in Human Prenatal Ontogenesis

Using light and electron microscopy and immunocytochemistry methods, structural organization of the formed blood-cerebrospinal fluid barrier (BCSFB) of the human brain choroid plexus in embryos of 6–9 weeks of development was studied. The main structures peculiar to the mature BCSFB have been established to appear with formation of the choroid plexus at the end of the 2nd month of the human intrauterine development. Fenestrae in the choroid plexus capillary endothelium are revealed since the 9th week of prenatal development. Characteristic of the human embryonic BCSFB are a poor development of the plexus capillary basal membrane, scanty pericytes, a high activity of interstitial macrophages, which suggests the barrier immaturity. A significant amount of cytoplasmic glycogen inclusions revealed in plexus epitheliocytes seems to be due to peculiar trophic requirements of developing brain cells under conditions of an insufficient development of the local blood supply.     

Growth Inhibitory Effect of Polyunsaturated Fatty Acids (PUFAs) on Colon Cancer Cells via Their Growth Inhibitory Metabolites and Fatty Acid Composition Changes

Background

Colorectal cancer is common. Polyunsaturated fatty acids (PUFAs) exert growth-inhibitory and pro-apoptotic effects on colon cancer cells. Metabolites of PUFAs such as prostaglandins (PGs), leukotrienes (LTs) and lipoxins (LXs) play a significant role in colon cancer.

Methods

Human colon cancer LoVo and RKO cells were cultured with different concentration of PUFAs and 5-fluorouracil (5-FU) in vitro. Cell morphological changes, fatty acid composition, formation of PGE2, LTB4 and LXA4 and expression of COX-2, ALOX5, PGD synthase (PGDS), microsomal prostaglandin E synthase (mPGES) were assessed in LoVo and RKO cells when supplemented with PUFAs and 5-FU.

Results

PUFAs and 5-FU inhibited growth of LoVo and RKO cells to the same extent at the doses used and produced significant alterations in their shape. As expected, higher concentrations of supplemented PUFAs were noted in the cells compared to control. LA, GLA, AA, ALA and EPA supplementation to LoVo cells suppressed production of PGE₂, LTB₄,and ALOX5, mPGES expression, but enhanced that of LXA₄; whereas DHA enhanced PGE₂ and LXA₄ synthesis but decreased LTB₄ formation and COX-2, ALOX5, mPGES expression. In contrast, 5-FU enhanced formation of PGE₂, LTB₄ and mPGES expression, but suppressed LXA₄ synthesis and COX-2 expression. PGE₂, LTB₄ synthesis and ALOX5 expression was suppressed by LA, GLA, ALA and DHA; whereas AA, EPA and 5-FU enhanced PGE₂ but paradoxically AA decreased and EPA and 5-FU enhanced LTB4 synthesis in RKO cells. All the PUFAs tested enhanced, while 5-FU decreased LXA₄ formation in RKO cells; whereas GLA, AA, and 5-FU augmented while LA, ALA, EPA and DHA enhanced COX-2 expression in RKO cells.

Conclusions

Tumoricidal action of PUFAs on colorectal LoVo and RKO cancer cells in vitro was associated with increased formation of LXA₄, decreased synthesis of PGE₂ and LTB₄ and suppressed expression of COX-2, ALOX5, mPGES, whereas 5-FU produced contrasting actions on these indices.     

VITAMIN B6 TRANSPORT IN THE CENTRAL NERVOUS SYSTEM: IN VITRO STUDIES

Abstract— The transport into and release of tritium labeled vitamin B₆ ([³H]B₆) from rabbit brain slices and isolated choroid plexuses were studied. In vitro, both brain slices and choroid plexus concentrated [³H]B₆ by an energy dependent uptake system when [³H]pyridoxine (PIN) was added to the incubation medium. Most of the [³H] within the tissues was phosphorylated [³H]B₆. In each tissue, the nonphosphorylated vitamers inhibited the uptake of [³H]PIN from the medium significantly more than the phosphorylated vitamers. The concentrations of the nonphosphorylated B₆ vitamers necessary to inhibit brain and choroid plexus uptake of [³H]PIN from the medium by 50% were approx 0.4 μm and 5–10μm respectively after a 30 min incubation. Both brain slices and choroid plexus readily released (46 and 56% respectively in 30 min) previously accumulated [³H]B₆ into artificial CSF. However, brain slices released only nonphosphorylated [³H]B₆, whereas the choroid plexus released predominantly phosphorylated [³H]B₆. Addition of unlabeled PIN to the release media significantly increased the percentage of [³H]B₆ released by both brain slices and choroid plexus. The results of these in vitro studies provide evidence that: (1) both brain slices and chloroid plexus possess specific uptake and release mechanisms for B₆, and (2) these mechanisms tend to regulate intracellular B₆ levels. These studies also suggest that the choroid plexus serves as a locus for the transfer of B₆ from blood to CSF and is the source of most of the phosphorylated B₆ in CSF.     

Recurrent ventricular fibrillation treated by multiple countershocks.

Dichlorphenamide was administered to 13 patients with chronic respiratory failure, and the effects on gas exchange at rest and during exercise and on the acid-base state of CSF were observed. The ventilation for a given level of CO₂ production was increased both at rest and during exercise, resulting in an increased arterial Po₂ and decreased Pco₂.The ventilatory stimulation paralleled the development of a metabolic acidosis but was not associated with tissue CO₂ accumulation. Indeed, CSF Pco₂ and the oxygenated mixed venous (rebreathing) Pco₂ fell by the same amount as arterial Pco₂. The level of CO₂ elimination after two minutes of exercise was as great for a given work load after dichlorphenamide as before. These findings do not support the view that the drug impairs CO₂ transport from tissues either at rest or during exercise. They are most consistent with the view that the primary locus of action of dichlorphenamide in therapeutic doses is the kidney. The metabolic acidosis which results is likely the basis of the respiratory stimulatin, perhaps by its effects on the CSF H₂CO₃-HCO₃ - system. Inhibition of carbonic anhydrase in the red cell and choroid plexus are probably unimportant effects.     

The blood-cerebrospinal fluid barrier is a major pathway of cerebral creatinine clearance: involvement of transporter-mediated process

There is still incomplete evidence for the cerebral clearance of creatinine (CTN) which is an endogenous convulsant and accumulates in the brain and CSF of patients with renal failure. The purpose of this study was to clarify the transporter-mediated CTN efflux transport from the brain/CSF. In vivo data demonstrated that CTN after intracerebral administration was not significantly eliminated from the brain across the blood-brain barrier. In contrast, the elimination clearance of CTN from the CSF was 60-fold greater than that of inulin, reflecting CSF bulk flow. Even in renal failure model rats, the increasing ratio of the CTN concentration in the CSF was lower than that in the plasma, suggesting a significant role for the CSF-to-blood efflux process. The inhibitory effects of inhibitors and antisense oligonucleotides on CTN uptake by isolated choroid plexus indicated the involvement of rat organic cation transporter 3 (rOCT3) and creatine transporter (CRT) in CTN transport. rOCT3- and CRT-mediated low-affinity CTN transport with K(m) values of 47.7 and 52.0 mM, respectively. Our findings suggest that CTN is eliminated from the CSF across the blood-CSF barrier as a major pathway of cerebral CTN clearance and transporter-mediated processes are involved in the CTN transport in the choroid plexus.     

Differential effect of chondroitin-4-sulfate on the immediate and delayed prostaglandin E2 release from osteoblasts

The present study examines the effect of chondroitin-4-sulfate (C4S) on the immediate (non-inflammatory conditions) and the delayed (inflammatory conditions) prostaglandin E₂ (PGE₂) release from rat calvarial osteoblasts. An immediate low release of PGE₂ was induced by PAF, phorbol ester and arachidonic acid but not by IL1β, TNF-α and LPS whereas a delayed high release of PGE₂ was induced by the inflammatory agents IL1β, TNF-α and LPS but not by PAF, phorbol ester and arachidonic acid. C4S had no effect on the immediate PGE₂ release but inhibited the delayed release of PGE₂. IL1β, TNF-α and LPS enhanced the expression of COX-2 and mPGES1 whereas phorbol ester enhanced COX-2 expression only. PAF and arachidonic acid had no effect on the expression of COX-2 and mPGES1. C4S inhibited the enhanced expression of COX-2 and mPGES1 but had no effect on the IL1β-induced decrease of I-κBα and nuclear translocation of NF-κB. These results indicate that the beneficial effects of C4S in bone inflammatory diseases might be due to a specific inhibition of the delayed high PGE₂ release from osteoblasts.     

The effect of adrenaline and Walker‐256 tumour‐induced cachexia upon Kupffer cell metabolism

Kupffer cells (KC), the liver macrophages, are able to produce PGE₂, which is involved in immune suppression and in the aggravation of cancer cachexia due to interference with lipid metabolism in the liver. Since tumour‐bearing (TB) rats present high plasma epinephrine levels, and this hormone is able to affect macrophage metabolism and function, we have assessed the effect of epinephrine (5 nm ) upon Kupffer cell PGE₂ production. Epinephrine induced increased production of PGE₂ both by control (3·5‐fold) and TB rats (27 per cent) KC, an effect blocked by propranolol. Enhancement of cAMP content in the cells by addition of isoproterenol (0·1 μm ) to the incubations, however, failed to induce the same response in the cells. Nevertheless, when phenylephrine (1 μm ) was added to the incubation, a similar pattern of PGE₂ production to that observed for epinephrine was found for control and TB rat KC. We propose that the effect of epinephrine upon KC PGE₂ production is mediated by α‐adrenergic receptors and that Ca²⁺ is involved in the response, since increasing concentrations of the ion added to the incubation medium (0·25, 0·5 and 1·0 mm ) enhanced the eicosanoid production, while EDTA abolished the response. Copyright © 1999 John Wiley & Sons, Ltd.     

NIACIN AND NIACINAMIDE TRANSPORT IN THE CENTRAL NERVOUS SYSTEM. IN VIVO STUDIES

Abstract– The concentration ol niacinamide in plasma and CSF was 0.5 and 0.7 μm respectively. The, mechanisms by which niacin and niacinamide, which are not synthesized in brain, enter brain, CSF and choroid plexus were investigated by injecting [¹⁴C]niacin or [¹⁴C]niacinamide intravenously and intraventricularly. [¹⁴C]Niacin or [¹⁴C]niacinamide, with or without unlabeled niacin or niacinamide, were infused intravenously at a constant rate into conscious rabbits. At 3 h, [¹⁴C]niacinamide, but not [¹⁴C]niacin, readily entered CSF, choroid plexus and brain. The addition of 4.1 mmol/kg niacinamide to the infusate markedly depressed the relative entry of [¹⁴C]niacinamide into choroid plexus and brain but not into CSF. After intraventricular injection, [¹⁴C]niacin was rapidly cleared from CSF and readily entered brain and choroid plexus. The addition of unlabeled niacin to the intraventricular injectate decreased the clearance of [¹⁴C]niacin from CSF and the entry of [¹⁴C]niacin into choroid plexus and brain. Unlike niacin, carrier niacinamide (82 μmol) in the injectate did not depress the extremely rapid clearance of intraventricularly injected [¹⁴C]niacinamide from CSF but did decrease the entry of [¹⁴C]niacinamide into brain. These results show that the control of entry and exit of niacinamide and niacin is the mechanism, at least in part, by which total niacin and NAD levels in brain cells are regulated. In the case of niacinamide which readily passes between CSF and plasma, the regulation of entry of niacinamide into brain cells by a high affinity accumulation system is an integral part of the homeostatic system. In the case of niacin, penetration into CSF and the extracellular space of brain from plasma as well as regulation of entry into brain cells by a saturable accumulation system are two distinct parts of the homeostatic system. In vivo, niacin that enters the central nervous system is converted to the principal plasma vitamer, niacinamide, in its free or bound forms such as NAD.