Transport of Leucine-Enkephalin Across the Blood-Brain Barrier in the Perfused Guinea Pig Brain

Transport of [tyrosyl-3,5-3H]enkephalin-(5-L-leucine) [( 3H]Leu-enkephalin) across the blood-brain barrier was studied in the adult guinea pig, by means of vascular perfusion of the head in vivo. The unidirectional transfer constant (Kin) estimated from the multiple-time uptake data for [3H]Leu-enkephalin ranged from 3.62 X 10(-3) to 3.63 X 10(-3) ml min-1 g-1 in the parietal cortex, caudate nucleus, and hippocampus. Transport of [3H]Leu-enkephalin was not inhibited by unlabelled L-tyrosine (the N-terminal amino acid) at a concentration as high as 5 mM, or by the inhibitor of aminopeptidase activity bacitracin (2 mM), suggesting that there was no enzymatic degradation of peptide at the blood-brain barrier. By contrast, 2 mM unlabelled Leu-enkephalin strongly inhibited the unidirectional blood-to-brain transport of [3H]Leu-enkephalin by 74-78% in the parietal cortex, caudate nucleus, and hippocampus. The tetrapeptide tyrosyl-glycyl-glycyl-phenylalanine (without the C-terminal leucine of Leu-enkephalin), at a concentration of 5 mM, caused a moderate inhibition ranging from 15 to 29% in the brain regions studied, whereas the tetrapeptide glycyl-glycyl-phenylalanyl-leucine (without the N-terminal tyrosine) at 5 mM was without effect on Leu-enkephalin transport. Unidirectional brain uptake of Leu-enkephalin was not altered in the presence of naloxone at a concentration as high as 3 mM (1 mg/ml), suggesting that there is no binding of Leu-enkephalin to opioid receptors at the blood-brain barrier. It is concluded that there is a specific transport mechanism for Leu-enkephalin at the blood-brain barrier in the guinea pig.(ABSTRACT TRUNCATED AT 250 WORDS)     

Saturable mechanism for delta sleep-inducing peptide (DSIP) at the blood-brain barrier of the vascularly perfused guinea pig brain

Cellular uptake of [125I] labelled DSIP at the luminal interface of the blood-brain barrier (BBB) was studied in the ipsilateral perfused in situ guinea pig forebrain. Regional unidirectional transfer constants (Kin) calculated from the multiple-time brain uptake analysis were 0.93, 1.33 and 1.66 microliter.min-1 g-1 for the parietal cortex, caudate nucleus and hippocampus, respectively. In the presence of 7 microM unlabelled DSIP the brain uptake of [125I]-DSIP (0.3 nM) was inhibited, the values of Kin being reduced to 0.23-0.38 microliter.min-1 g-1, values that were comparable with the Kin for mannitol. The rapidly equilibrating space of brain, measured from the intercept of the line describing brain uptake versus time on the brain uptake ordinate, Vi, was greater for [125I]-DSIP than for mannitol; in the presence of unlabelled DSIP this was reduced to that of mannitol, and it was suggested that the larger volume for [125I]-DSIP represented binding at specific sites on the brain capillary membrane. L-tryptophan, the N-terminal residue of DSIP, in concentrations of 7 microM and 1 mM, inhibited Kin without affecting Vi. A moderate inhibition of Kin was obtained by vasopressin ([Arg8]-VP), but only at a concentration as high as 0.2 mM. The results suggest the presence of a high affinity saturable mechanism for transport of DSIP across the blood-brain barrier, with subsequent uptake at brain sites that are highly sensitive to L-tryptophan, and may be modulated by [Arg8]-VP.     

Selective inhibition of [3H]nitrendipine binding to brain and cardiac membranes by bacitracin

The effects of bacitracin were investigated on [3H]nitrendipine binding to rat brain and cardiac membranes in a low ionic strength (5 mM Tris-HCl) buffer. Bacitracin inhibited [3H]nitrendipine binding to rat brain and cardiac membranes with IC50 values of 400 +/- 100 and 4600 +/- 400 micrograms/mL, respectively. Scatchard analysis in brain membranes revealed that bacitracin inhibited [3H]nitrendipine binding primarily by reducing the Bmax but also by producing a small increase in the Kd. In brain membranes, Na+ (100 mM) and Ca2+ (2 mM) reduced the potency of bacitracin to inhibit [3H]nitrendipine binding by approximately sixfold with IC50 values of 2600 +/- 300 and 2100 +/- 400 micrograms/mL observed for bacitracin in the presence of 100 mM Na+ and 2 mM Ca2+, respectively. The EC50 values for the effects of Na+ and Ca2+ were 800 +/- 200 microM and 25 +/- 5 mM. K+, Mg2+, choline, and increasing the assay buffer of Tris-HCl to 50 mM also decreased the inhibition of [3H]nitrendipine binding by bacitracin. These results suggest that bacitracin specifically modulates [3H]nitrendipine binding in a cation-dependent manner and that brain and cardiac dihydropyridine binding sites are either biochemically different or exist in a different membrane environment.     

Pantothenic Acid Transport Through the Blood-Brain Barrier

The unidirectional influx of D-pantothenic acid (PA) across cerebral capillaries, the anatomical locus of the blood-brain barrier, was measured with an in situ rat brain perfusion technique using [3H]D-PA (1.1 Ci/mmol). PA was transported across the blood-brain barrier by a saturable system that could be described by a Michaelis-Menten transport model with a half-saturation concentration and maximal influx rate of 19 microM and 0.21 nmol/g of brain/min, respectively. PA (0.3 microM) transport through the blood-brain barrier was significantly inhibited by probenecid, nonanoic acid, and biotin (all less than or equal to 0.25 mM), but not by penicillin G, pyruvate, beta-hydroxybutyrate, L-leucine (all 1 mM), or poly-L-lysine HBr (1 mg/ml). Probenecid (0.25 mM), nonanoic acid (0.5 mM), and PA (1.0 mM) did not inhibit [3H]L-leucine transport through the blood-brain barrier, whereas 30 microM-L-leucine inhibited [3H]leucine transport to 23% of control values. Thus, PA is transported through the blood-brain barrier by a low-capacity, saturable transport system with a half-saturation concentration approximately 10 times the plasma PA concentration. Although involved in the transfer of PA from blood into brain, this system does not play an important regulatory role in the synthesis of CoA from PA in brain.     

Studies of angiotensin I converting enzyme: effects of kinins, bacitracin, gamma-aminobutyric and epsilon-aminocaproic acids, and related compounds on substrate binding and catalysis in vitro

Bradykinin and 22 of its analogs were evaluated for their abilities to inhibit the hydrolysis of [3H]hippurylglycylglycine by purified porcine kidney angiotensin I converting enzyme. The mean inhibitory concentration (IC50) for bradykinin was 1.2 +/- 0.2 X 10(-6) M. Except for Ile-Ser-bradykinin and [Sar4]-bradykinin, none of the kinin analogs were more potent in this regard than bradykinin. Bacitracin, gamma-aminobutyric acid, epsilon-aminocaproic acid, and structurally related compounds were also tested. The IC50 value for bacitracin was 1.9 +/- 0.4 X 10(-4) M, gamma-aminobutyric acid, 83.4 +/- 7.2 mM, and for epsilon-aminocaproic acid, 7.0 +/- 1.4 mM. Compounds were also evaluated for their abilities to prevent 125I-labelled [Tyr1]-kallidin binding to angiotensin I converting enzyme inhibited by EDTA. The IC50 values for bradykinin, bacitracin, gamma-aminobutyric acid, and epsilon-aminocaproic acid were 1.6 +/- 0.3 X 10(-8) M, 2.6 +/- 0.9 X 10(-6) M, greater than 291 mM, and 13.2 +/- 3.9 mM, respectively.     

Hypoxanthine transport through the blood-brain barrier

The unidirectional influx of hypoxanthine across cerebral capillaries, the anatomical locus of the blood=brain barrier, was measured with an in situ rat brain perfusion technique employing [³H]hypoxanthine. Hypoxanthine was transported across the blood-brain barrier by a saturable system with a one-half saturation concentration of approximately 0.4 mM. The permeability-surface area product was 3×10^–4 sec^–1 with a hypoxanthine concentration of 0.02 M in the perfusate. Adenine (4 mM) and uracil and theophylline (both 10 mM), but not inosine (10 mM) or leucine (1 mM), inhibited hypoxanthine transfer through the blood-brain barrier. Thus, hypoxanthine is transported through the blood-brain barrier by a high-capacity, saturable transport system with a half-saturation concentration about 100 times the plasma hypoxanthine concentration. Although involved in the transport hypoxanthine from blood into brain, this system is not powerful enough to transfer important quantities of hypoxanthine from blood into brain.     

Biotin Transport Through the Blood-Brain Barrier

The unidirectional influx of biotin across cerebral capillaries, the anatomical locus of the blood-brain barrier, was measured with an in situ rat brain perfusion technique employing [3H]biotin. Biotin was transported across the blood-brain barrier by a saturable system with a one-half saturation concentration of approximately 100 microM. The permeability-surface area products were 10(-4) s-1 with a biotin concentration of 0.02 microM in the perfusate. Probenecid, pantothenic acid, and nonanoic acid but not biocytin or biotin methylester (all 250 microM) inhibited biotin transfer through the blood-brain barrier. The isolated rabbit choroid plexus was unable to concentrate [3H]biotin from medium containing 1 nM [3H]biotin. These observations provide evidence that: biotin is transported through the blood-brain barrier by a saturable transport system that depends on a free carboxylic acid group, and the choroid plexus is probably not involved in the transfer of biotin between blood and cerebrospinal fluid.     

Effect of thyrotropin-releasing hormone on the carbohydrate structure of secreted mouse thyrotropin. Analysis by lectin affinity chromatography

Thyrotropin (TSH) is a glycoprotein hormone whose secretion from the anterior pituitary is regulated, in part, by the hypothalamic tripeptide thyrotropin-releasing hormone (TRH). We have used serial lectin affinity analysis to explore whether TRH, in addition to promoting TSH secretion, alters the carbohydrate structure of secreted TSH. Hypothyroid mouse hemipituitaries were incubated in medium containing [3H] mannose, [3H]glucosamine, or [3H]fucose either with or without 10(-7) M TRH. TSH was immunoprecipitated, proteolytically digested into glycopeptides, and chromatographed on serial lectin-Sepharose columns. Under basal conditions, 37% of secreted [3H]mannose-labeled TSH glycopeptides failed to bind to concanavalin A (ConA)-Sepharose, 55% bound and eluted with 10 mM alpha-methylglucoside, and 8% bound and eluted with 500 mM alpha-methylmannoside. Approximately 35% of glycopeptides not binding to ConA-Sepharose were bound by pea lectin-Sepharose, suggesting the presence of certain core fucosylated triantennary complex oligosaccharides. TRH caused a 2-fold increase in secretion of [3H]mannose-labeled TSH glycopeptides due almost exclusively to a specific increase in structures that bound to ConA-Sepharose and eluted with 10mM alpha-methylglucoside, corresponding to biantennary complex or unusual hybrid species. There was no change in the distribution of intrapituitary TSH glycopeptides with TRH. Acid hydrolysis of secreted proteins showed little metabolism of the tritiated sugar precursors, except for a 20% conversion of [3H]mannose to [3H]fucose. Moreover, ConA-Sepharose chromatography of secreted [3H]glucosamine- and [3H]fucose-labeled TSH glycopeptides showed similar increases in ConA-Sepharose binding with TRH as noted with [3H]mannose labeling. Subsequent lectin analysis of secreted [3H] mannose-labeled TSH glycopeptides on erythroagglutinating phytohemagglutinin-Sepharose and leukoagglutinating phytohemagglutinin-Sepharose disclosed no significant differences in TRH-treated versus control samples. These data suggest that secreted mouse TSH has greater carbohydrate heterogeneity than has been recognized previously. In addition, TRH in vitro promotes the secretion of specific TSH molecules apparently enriched in biantennary complex or unusual hybrid oligosaccharides.     

Brain receptor binding characteristics and pharmacokinetics of JTP-2942, a novel thyrotropin-releasing hormone (TRH) analogue

JTP-2942 competed with [3H]-Me-TRH for the binding sites in rat brain in vitro, and its inhibitory effect was approximately 17 times less potent than TRH, as shown by Ki values of 673 and 39.7 nM, respectively. Both JTP-2942 and TRH significantly increased apparent dissociation constant (Kd values) for brain [3H]-Me-TRH binding. Intravenous injection of JTP-2942 (0.3-3 mg/kg) and TRH (3 and 10 mg/kg) produced a significant reduction of [3H]-Me-TRH binding sites (Bmax values) in rat brain. Although the decrease by TRH was maximal 10 min after the injection and declined rapidly with time, the decrease by JTP-2942 (1 and 3 mg/kg) tended to be maximal at 30 min later and it lasted until 120 min. The intravenous injection of JTP-2942 was at least 3 times more potent than that of TRH in decreasing Bmax values for brain [3H]-Me-TRH binding. Plasma concentration of JTP-2942 (0.3-3 mg/kg) after intravenous injection in rats rose with the increase of dose, and it peaked immediately after the injection, thereafter decreasing with t1/2 of 19.3-29.9 min. It is concluded that JTP-2942, compared to TRH, may exert fairly potent and sustained occupation of brain TRH receptors under in vivo condition. Thus, JTP-2942 could be clinically useful for the treatment of CNS disorders.     

Chlordiazepoxide is a competitive thyrotropin-releasing hormone receptor antagonist in GH3 pituitary tumour cells

Addition of thyrotropin-releasing hormone (TRH) to [3H]-inositol pre-labelled GH3 pituitary tumour cells suspended in medium containing 10mM lithium chloride led to a rapid diminution in cellular [3H]-inositol and increase in [3H]-inositol 1-phosphate (InslP), [3H]-inositol bisphosphate (InsP2) and [3H]-inositol trisphosphate (InsP3). In the presence of the benzodiazepine tranquillizer, chlordiazepoxide, the TRH concentration-response curves for these effects were shifted to the right in a parallel fashion. The Ki for chlordiazepoxide in inhibiting all four responses was 1.5 X 10(-5)M. Chlordiazepoxide did not inhibit the small bombesin-induced rise in [3H]-InslP. Another benzodiazepine, diazepam, was less active. The TRH-induced rise in cytosolic free calcium monitored in Quin-2-loaded GH3 cells was also blocked by chlordiazepoxide in a competitive manner, while that induced by high K+-induced depolarisation was unaffected. It is suggested that chlordiazepoxide acts as a competitive antagonist at the level of the TRH receptor.     

Myo-inositol transport through the blood-brain barrier

The unidirectional transport of [³H]myo-inositol across cerebral capillaries, the anatomical locus of the blood-brain barrier, was measured using an in situ rat brain perfusion technique. Myo-inositol was transported across the blood-brain barrier by a low capacity, saturable system with a one-half saturation concentration of 0.1 mM. The permeability surface-area product was 6.2×10^–5S^–1 with a myo-inositol concentration of 0.02 mM in the perfusate. The myo-inositol stereoisomer scyllo-inositol but not (+)-chiro-inositol (both 1 mM) inhibited myo-inositol transfer through the blood-brain barrier. These observations provide evidence that myo-inositol is transferred through the blood-brain barrier by simple diffusion and a stereospecific, saturable transport system.     

Measurement of Solute Transport Across the Blood–Brain Barrier in the Perfused Guinea Pig Brain: Method and Application to N-Methyl-α-Aminoisobutyric Acid

A technique for the vascular perfusion of the guinea pig head in vivo, suitable for measurements of blood-to-brain transport under controlled conditions of arterial inflow, has been developed. With a perfusion pressure ranging between 13 and 18 kPa and PCO2 in the arterial inflow of 5 and 5.5 kPa, cerebral blood flow, measured with [14C]butanol, was about 1 ml min-1 g-1 in the cerebral cortex, hippocampus, and caudate-putamen of the ipsilateral hemisphere; in the cerebellum and pontine white matter it was considerably less, and much higher perfusion pressures were required to establish equal blood flow throughout the whole brain. Regional water content, Na+/K+ ratio, ATP, energy charge potential, and lactate content of the ipsilateral side of perfused and nonperfused brain were not significantly different after 10 min perfusion. The D-[3H]mannitol space did not exceed 1% after 30 min of perfusion, indicating the integrity of the barrier. Over this period, EEG, ECG, and respiratory waveform remained normal. When [14C]N-methyl-alpha-aminoisobutyric acid (MeAIB), and D-[3H]mannitol were perfused together over periods extending to 30 min progressive uptakes of both solutes by the parietal cortex could be measured, and the unidirectional transfer constants estimated from multiple time-uptake data. The Kin for MeAIB (0.75 X 10(-3) ml min-1 g-1) was some three times that for mannitol. It is concluded that the technique provides a stable, well-controlled environment in the cerebral microvasculature of the ipsilateral perfused brain hemisphere suitable for examining the transport of slowly penetrating solutes into the brain.     

Effect of Dexamethasone on Transport of α-Aminoisobutyric Acid and Sucrose Across the Blood-Brain Barrier

The effect of glucocorticoids on the blood-brain barrier (BBB) was studied in rats following a single injection or 3 days of dexamethasone administration. Tracers with a low permeability across the intact endothelium, [14C]sucrose and alpha-[3H]aminoisobutyric acid ([3H]AIB), were simultaneously injected intravenously in untreated rats or in rats treated with dexamethasone. Unidirectional blood-to-brain transfer constants (Ki) in 14 regions of the rat brain were determined. In regions of control brain, average Ki values for AIB and sucrose were approximately 0.0020 and 0.00060 ml g-1 min-1, respectively. The lowest transfer constants were found in caudate nucleus, hippocampus, white matter, and cerebellum. In dexamethasone-treated animals, Ki values for both sucrose and AIB markedly decreased by 30-50% in almost all brain regions. These results indicate that a single injection or 3 days of treatment with dexamethasone causes an apparent reduction in the normal BBB permeability, and dexamethasone may greatly interfere with drug delivery into brain. These observations may have an importance for the administration of drugs in brain disease in the presence of steroids.     

Thyrotropin-releasing hormone (TRH) and its analog (DN-1417): interaction with pentobarbital in choline uptake and acetylcholine synthesis of rat brain slices

TRH or its analog DN-1417 (gamma-butyrolactone-gamma-carbonyl-L-histidyl-L-proliamide) given 15 min after intravenous (i.v.) administration of pentobarbital (30 mg/kg) markedly shortened the pentobarbital-induced sleeping time in rats. This effect was almost completely abolished by intracerebroventricular pretreatment with atropine methylbromide (20 micrograms/rat), thereby suggesting the involvement of cholinergic mechanism. The action mechanism was investigated using rat brain slices. TRH (10(-6)-10(-4)M) or DN-1417 (10(-7)-10(-5)M) caused significant increases in the uptake of [3H]-choline into striatal slices. TRH(10(-4)M) or DN-1417(10(-5)M) also stimulated the conversion of [3H]-choline to [3H]-acetylcholine in striatal slices. A 30% reduction of acetylcholine synthesis from [3H]-choline in hippocampal slices and a 40% reduction of [3H]-choline uptake in slices of cerebral cortex, hippocampus and hypothalamus were observed in rats pretreated with pentobarbital (60 mg/kg, i.v.). TRH or DN-1417 (20 mg/kg, i.v.) given 15 min after the administration of pentobarbital markedly reversed both of the pentobarbital effects. Direct application of pentobarbital (5 X 10(-4)M) to slices in vitro also caused a 20-40% reduction of [3H]-choline uptake of cerebral cortex, hippocampus and diencephalon. A concomitant application of TRH(10(-4)M) or DN-1417(10(-5)M) and pentobarbital abolished the pentobarbital effect. These results provide neurochemical evidence that the antagonistic effects of TRH and DN-1417 on pentobarbital-induced narcosis are closely related to alterations in the rat brain choline uptake and acetylcholine synthesis, which are considered to be measures of the activity of cholinergic neurons.     

4-Aminobutyraldehyde as a precursor convertible to gamma-aminobutyric acid in vivo

It was found that 4-aminobutyraldehyde (ABAL) is a precursor convertible to gamma-aminobutyric acid (GABA) in vivo. [2,3-3H]ABAL was synthesized from [2,3-3H]putrescine. After the subcutaneous administration of [3H]ABAL at the dose of 1 mumol/g body weight, [3H]GABA was produced in the mouse brain in an amount of about 350 nmol/g brain in 10 min. After oral administration of [3H]ABAL at the dose of 2 mumol/g body weight, [3H]GABA was also produced in the brain in an amount of about 530 nmol/g brain in 30 min. It seems that peripherally administered ABAL penetrates the blood-brain barrier into the central nervous system and is rapidly metabolized to GABA in the brain.     

Brain-to-blood efflux transport of estrone-3-sulfate at the blood-brain barrier in rats

Efflux transport of estrogens such as estrone-3-sulfate (E1S), and estrone (E1) across the blood-brain barrier (BBB) was evaluated using the Brain Efflux Index (BEI) method. The apparent BBB efflux rate constant (Keff) of [3H]E1S, and [3H]E1 was 6.63 x 10(-2) +/- 0.77 x 10(-2) min(-1), and 6.91 x 10(-2) +/- 1.23 x 10(-2) min(-1), respectively. The efflux transport of [3H]E1S from brain across the BBB was a saturable process with Michaelis constant (Km) of 96.0 +/- 34.4 microM and 93.4 +/- 22.0 microM estimated by two different methods. By determining [3H]E1S metabolites using high performance liquid chromatography (HPLC) after intracerebral injection, significant amounts of [3H]E1S were found in the jugular venous plasma, providing direct evidence that most of [3H]E1S is transported from brain across the BBB in intact form. To compare the apparent efflux clearance across the BBB of E1S with that of E1, the brain distribution volume of E1S and E1 was estimated using the brain slice uptake method. The apparent efflux clearance of [3H]E1S was determined to be 74.9 +/- 3.8 microl/(min x g brain) due to the distribution volume of 1.13 +/- 0.06 ml/g brain. By contrast, the apparent efflux clearance of E1 was more than 227 +/- 3 microl/(min x g brain), since the distribution volume of [3H]E1 at 60 min was 3.28 +/- 0.13 ml/g. The E1S efflux transport process was inhibited by more than 40% by coadministration of bile acids (taurocholate, and cholate), and organic anions (sulfobromophthalein, and probenecid), whereas other organic anions did not affect the E1S efflux transport. The [3H]E1S efflux was significantly reduced by 48.6% after preadministration of 5 mM dehydroepiandrosterone sulfate. These results suggest that E1S is transported from brain to the circulating blood across the BBB via a carrier-mediated efflux transport system.     

A D-Glucose-Sensitive Cytochalasin B Binding Component of Cerebral Microvessels

The technique of photoaffinity labelling with [4-3H]cytochalasin B was applied to osmotically lysed cerebral microvessels isolated from sheep brain. Cytochalasin B was photo-incorporated into a membrane protein of average apparent Mr 53,000. Incorporation of cytochalasin B was inhibited by D-glucose, but not by L-glucose, which strongly suggests that the labelled protein is, or is a component of, the glucose transporter of the blood-brain barrier. Investigation of noncovalent [4-3H]cytochalasin B binding to cerebral microvessels by equilibrium dialysis indicated the presence of a single set of high-affinity binding sites with an association constant of 9.8 +/- 1.7 (SE) microM-1. This noncovalent binding was inhibited by D-glucose, with a Ki of 23 mM. These results provide preliminary identification of the glucose transporter of the ovine blood-brain barrier, and reveal both structural and functional similarities to the glucose transport protein of the human erythrocyte.     

Functional relevance of carnitine transporter OCTN2 to brain distribution of l-carnitine and acetyl-l-carnitine across the blood–brain barrier

Transport of L-[3H]carnitine and acetyl-L-[3H]carnitine at the blood-brain barrier (BBB) was examined by using in vivo and in vitro models. In vivo brain uptake of acetyl-L-[3H]carnitine, determined by a rat brain perfusion technique, was decreased in the presence of unlabeled acetyl-L-carnitine and in the absence of sodium ions. Similar transport properties for L-[3H]carnitine and/or acetyl-L-[3H]carnitine were observed in primary cultured brain capillary endothelial cells (BCECs) of rat, mouse, human, porcine and bovine, and immortalized rat BCECs, RBEC1. Uptakes of L-[3H]carnitine and acetyl-L-[3H]carnitine by RBEC1 were sodium ion-dependent, saturable with K(m) values of 33.1 +/- 11.4 microM and 31.3 +/- 11.6 microM, respectively, and inhibited by carnitine analogs. These transport properties are consistent with those of carnitine transport by OCTN2. OCTN2 was confirmed to be expressed in rat and human BCECs by an RT-PCR method. Furthermore, the uptake of acetyl-L-[3H]carnitine by the BCECs of juvenile visceral steatosis (jvs) mouse, in which OCTN2 is functionally defective owing to a genetical missense mutation of one amino acid residue, was reduced. The brain distributions of L-[3H]carnitine and acetyl-L-[3H]carnitine in jvs mice were slightly lower than those of wild-type mice at 4 h after intravenous administration. These results suggest that OCTN2 is involved in transport of L-carnitine and acetyl-L-carnitine from the circulating blood to the brain across the BBB.     

Niacinamide transport through the blood-brain barrier

The unidirectional influx of niacinamide across cerebral capillaries, the anatomical locus of the blood-brain barrier, was measured with an in situ rat brain perfusion technique employing [¹⁴C]niacinamide. Niacinamide was transported rapidly across the blood-brain barrier by a system that was not saturable with 10 mM niacinamide in the perfusate. However, with periods of perfusion longer than 30 seconds, there was substantial backflow of [¹⁴C]niacinamide into the perfusate. Niacinamide (1.7 M) transport through the blood-brain barrier was not significantly inhibited by 3-acetylpyridine. Thus, niacinamide is transported rapidly and bidirectionally through the blood-brain barrier by a high capacity transport system. Although involved in the transfer of niacinamide between blood and brain, this transport system does not play an important regulatory role in the synthesis of NMN, NAD, and NADP from niacinamide in brain.     

Analysis by base exchange of thyrotropin-releasing hormone responsive and unresponsive inositol lipid pools in rat pituitary tumor cells

We have shown that there is an inositol (Ins) lipid pool in cloned rat pituitary tumor (GH3) cells that is hydrolyzed in response to thyrotropin-releasing hormone (TRH) and an unresponsive pool. Because others have suggested that incorporation of [3H]Ins by base exchange may not occur uniformly into Ins lipids in other cell types, we established conditions using permeabilized cells under which labeling occurs by Ins-phosphatidylinositol (PI) exchange in the absence of de novo PI synthesis to further characterize these pools in GH3 cells. In permeabilized cells incubated in buffer containing 10 mM Mg2+ and 0.1 mM CMP, [3H]Ins incorporation into lipids occurred by base exchange only. This was so because: 1) [3H]Ins incorporation into lipids displayed properties similar to that for release of 3H-labeled Ins by unlabeled Ins from PI in cells prelabeled in situ prior to permeabilization; and 2) there was no change in PI mass under these conditions. In permeabilized cells incubated in buffer with 0.1 mM [3H]Ins for 60 min, incorporation was 0.61 +/- 0.05 nmol of [3H]Ins/10(6) permeabilized cells, which amounted to 35% of PI, while the level of PI, measured as nonradioactive phosphorus, was 94 +/- 8.0% of control. Permeabilized GH3 cells were responsive to TRH. In cells prelabeled in situ and then permeabilized, TRH stimulated an increase in 3H-labeled Ins phosphates (IPs) in 20 min which was 10% of 3H radioactivity initially present in lipids. This increase in 3H-labeled IPs was 6.3 times the 3H radioactivity present in phosphatidylinositol 4,5-bisphosphate prior to stimulation. When prelabeled cells were exchanged with unlabeled Ins after permeabilization there was only a 10-16% decrease in 3H-labeled IP accumulation stimulated by TRH even though 3H-labeled lipids decreased to 52% of control. TRH did not affect labeling by [3H]Ins-PI exchange. In cells labeled by base exchange after permeabilization TRH stimulated a very small increase in 3H-labeled IPs of only 0.21 +/- 0.02% of 3H-labeled lipids in 20 min or only 7% of the 3H radioactivity in phosphatidylinositol 4,5-bisphosphate. These data show that in permeabilized GH3 cells base exchange can occur in the absence of de novo PI synthesis and that lipids that are preferentially labeled by base exchange comprise a pool that is less responsive to TRH than total Ins lipids.