Gamma-glutamyl transpeptidase does not act as a cystine transporter in brain microvessels

Gamma-glutamyl transpeptidase (GGTP) is highly enriched in blood-brain barrier (BBB) microvessels. According to the most cited hypothesis its functional role is amino acid transport across the BBB. To test this hypothesis the influence of GGTP inhibition on cystine uptake was measured in isolated brain microvessels. Adult porcine brain microvessels were enzymatically isolated, resulting in an enrichment of GGTP from 3 to 85 U/mg protein. The inhibitors 0.1 mM AT-125 combined with 20 mM hippurate reduced the GGPT enzyme activity by more than 98%. However this inhibition did not influence the uptake of [35S]-cystine, which is the substrate with the highest affinity in the GGTP-reaction. Instead increased glutathione (GSH) levels and elevated [35S] release were found. These results show that GGTP does not mediate the transport of cystine into brain microvessels in vitro and suggest that GGTP plays a role in cellular GSH metabolism.     

Effects of hormones on protein and amino acid metabolism in mammary-gland explants of mice

The effects of insulin, cortisol and prolactin on amino acid uptake and protein biosynthesis were determined in mammary-gland explants from mid-pregnant mice. Insulin stimulated [3H]leucine incorporation into protein within 15 min of adding insulin to the incubation medium. Insulin also had a rapid stimulatory effect on the rate of aminoiso[14C]butyric acid uptake, but it had no effect on the intracellular accumulation of [3H]leucine. Cortisol inhibited the rate of [3H]leucine incorporation into protein during the initial 4h of incubation, but it had no effect at subsequent times. [3H]Leucine uptake was unaffected by cortisol, but amino[14C]isobutyric acid uptake was inhibited after a 4h exposure period to this hormone. Prolactin stimulated the rate of [3H]leucine incorporation into protein when tissues were exposed to this hormone for 4h or more; up to 4h, however, no effect of prolactin was detected. At all times tested, prolactin had no effect on the uptake of either amino[14C]isobutyric acid or [3H]leucine. Incubation with actinomycin D abolished the prolactin stimulation of protein biosynthesis, but this antibiotic did not affect the insulin response. A distinct difference in the mechanism of action of these hormones on protein biosynthesis in the mammary gland is thus apparent.     

Proton-cotransport of pravastatin across intestinal brush-border membrane

PURPOSE: The purpose of the present study is to clarify the intestinal brush-border transport mechanism of a weak organic acid, pravastatin, an HMG-CoA reductase inhibitor. METHODS: The transport of pravastatin was studied by using intestinal brush-border membrane vesicles prepared from rabbit jejunum, and uptake by the membrane vesicles was measured using rapid filtration technique. RESULTS: The initial uptake of [14C]pravastatin was markedly increased with decreases in extravesicular pH and showed a clear overshoot phenomenon in the presence of a proton gradient (pHin/out = 7.5/5.5). A protonophore, carbonylcyanide p-trifluoromethoxyphenylhydrazone, significantly reduced the uptake of [14C]pravastatin. In addition, an ionophore for sodium, potassium and proton, nigericin, stimulated the uptake of [14C]pravastatin in the presence of a potassium gradient ([K+]in/[K+]out = 0/145 mM). On the other hand, neither the imposition of an inwardly directed sodium gradient nor an outwardly directed bicarbonate gradient stimulated the uptake of [14C]pravastatin. In the presence of a proton gradient (pHin/out = 7.5/5.5), the initial uptake of pravastatin was saturable with the apparent Kt of 15.2 +/- 3.2 mM and Jmax of 10.6 +/- 1.21 nmol/mg protein/10 sec. The uptake of pravastatin was significantly inhibited by monocarboxylic acid compounds such as acetic acid and nicotinic acid in a competitive manner but not by di- or tricarboxylic acids, or acidic amino acid. CONCLUSION: It was concluded that a pH-dependent transport of pravastatin across the brush-border membrane occurs by a proton-gradient dependent carrier-mediated mechanism rather than by simple diffusion of its unionized form.     

ATP-sensitive potassium channels are not expressed in brain microvessels

We used [3H]glibenclamide binding to assess ATP-sensitive K+ channels in isolated cerebral microvessels and in the cerebral cortex of the rat. We found no measurable specific glibenclamide binding in cerebral microvessels despite its abundance in cerebral cortical membranes, implying that ATP-sensitive K+ channels are not present in cerebral microvessels.     

Hepatic sinusoidal membrane transport of anionic drugs mediated by anion transporter Npt1

The purpose of our study was to establish the localization of the anion transporter Npt1 in liver and the relevance of Npt1 to carrier-mediated hepatic transport of beta-lactam antibiotics. Immunocytochemical examination of mouse liver with antiserum for Npt1 showed basolateral (sinusoidal) membrane localization. Function of Npt1 was characterized in Xenopus laevis oocytes. Injection of in vitro-transcribed cRNA into oocytes resulted in an increased uptake of [14C]benzylpenicillin (PCG). The Npt1-mediated uptake was saturable with a Michaelis constant (Km) of 0.46 +/- 0.18 mM and a maximum rate (Vmax) of 46.6 +/- 8.5 pmol/60 min/oocyte, and the uptake of [14C]PCG was independent of Na+ and pH, but dependent on chloride ion. Npt1-mediated [14C]PCG uptake was inhibited by several beta-lactam antibiotics and probenecid. Oocytes injected with Npt1-cRNA demonstrated significantly enhanced transport activity for other anionic compounds such as [14C]faropenem, [14C]foscarnet and [3H]mevalonic acid, as well as [14C]PCG, compared with water-injected oocytes. In conclusion, Npt1 is suggested to participate in hepatic sinusoidal membrane transport of organic anions such as beta-lactam antibiotics as well as inorganic anions for the efflux from hepatocyte-to-blood direction.     

Blood flow and uptake of glucose and amino acids in ischemic muscle

In order to examine muscle ischemia, muscle blood flow in the rat hindlimb was decreased by vessel ligation. Amino acid uptake, studied with [14C]alpha-aminoisobutyric acid, was decreased in ischemic Type I (soleus) muscle. Glucose uptake, studied with [14C]deoxyglucose, was increased in Type I muscle. These changes were temporally associated with histologic changes of ischemia in soleus muscle. Denervation, atrophy, and hypertrophy also produced uptake changes with these techniques, and although more prominent in soleus, the changes were also seen in Type II muscle.     

Biotransformation of 17-alkylsteroids in the equine: gas chromatographic-mass spectral identification of ten intermediate metabolites of methyltestosterone

Parenterally administered domoic acid, a structural analog of the excitatory amino acids glutamic acid and kainic acid, has specific effects on brain histology in rats, as measured using different anatomic markers. Domoic acid-induced convulsions affects limbic structures such as hippocampus and entorhinal cortex, and different anatomic markers can detect these neurotoxic effects to varying degrees. Here we report effects of domoic acid administration on quantitative indicators of brain metabolism and gliosis. Domoic acid, 2.25 mg/kg i.p., caused stereotyped behavior and convulsions in approximately 60% of rats which received it. Six to eight days after domoic acid or vehicle administration, the animals were processed to measure regional brain incorporation of the long-chain fatty acids [1-(14)C]arachidonic acid ([14C]AA) and [9,10-(3)H]palmitic acid ([3H]PA), or regional cerebral glucose utilization (rCMRglc) using 2-[1-(14)C]deoxy-D-glucose, by quantitative autoradiography. Others rats were processed to measure brain glial fibrillary acidic protein (GFAP) by enzyme-linked immunosorbent assay. Domoic acid increased GFAP in the anterior portion of cerebral cortex, the caudate putamen and thalamus compared with vehicle. However, in rats that convulsed after domoic acid GFAP was significantly increased throughout the cerebral cortex, as well as in the hippocampus, septum, caudate putamen, and thalamus. Domoic acid, in the absence of convulsions, decreased relative [14C]AA incorporation in the claustrum and pyramidal cell layer of the hippocampus compared with vehicle-injected controls. In the presence of convulsions, relative [14C]AA incorporation was decreased in hippocampus regions CA1 and CA2. Uptake of [3H]PA into brain was unaffected. Relative rCMRglc decreased in entorhinal cortex following domoic acid administration with or without convulsions. These results suggest that acute domoic acid exposure affects discrete brain circuits by inducing convulsions, and that domoic acid-induced convulsions cause chronic effects on brain function that are reflected in altered fatty acid metabolism and gliosis.     

Potential use of cytokine therapy in poultry

The time course of incorporation of [14C]arachidonic acid and [3H]docosahexaenoic acid into various lipid fractions in placental choriocarcinoma (BeWo) cells was investigated. BeWo cells were found to rapidly incorporate exogenous [14C]arachidonic acid and [3H] docosahexaenoic acid into the total cellular lipid pool. The extent of docosahexaenoic acid esterification was more rapid than for arachidonic acid, although this difference abated with time to leave only a small percentage of the fatty acids in their unesterified form. Furthermore, uptake was found to be saturable. In the cellular lipids these fatty acids were mainly esterified into the phospholipid (PL) and the triacyglycerol (TAG) fractions. Smaller amounts were also detected in the diacylglycerol and cholesterol ester fractions. Almost 60% of the total amount of [3H]Docosahexaenoic acid taken up by the cells was esterified into TAG whereas 37% was in PL fractions. For arachidonic acid the reverse was true, 60% of the total uptake was incorporated into PL fractions whereas less than 35% was in TAG. Marked differences were also found in the distribution of the fatty acids into individual phospholipid classes. The higher incorporation of docosahexaenoic acid and arachidonic acid was found in PC and PE, respectively. The greater cellular uptake of docosahexaenoic acid and its preferential incorporation in TAG suggests that both uptake and transport modes of this fatty acid by the placenta to fetus is different from that of arachidonic acid.     

Decreased uptake of 14C-labeled dicarboxylic amino acids in rapidly growing hepatomas

In contrast to the increased uptake of amino acids which has been found in many neoplastic cells, we have observed a decrease in the net uptake of [14C]aspartate and [14C]glutamate in rapidly growing hepatomas relative to rat host liver. When measured 10 min after s.c. injection, the radioactivity from 14C-labeled dicarboxylic amino acids was greater in liver than in all other tissues examined (blood, skeletal, muscle, heart, spleen, lung, and brain) except kidney, where there was an approximately 2-fold greater uptake of aspartate and 10-fold greater uptake of glutamate. Mean uptakes in the rapidly growing Morris hepatomas 7288CTC and 7777 were 19 to 26% of corresponding values for the host livers. Comparison with uptake of 3H2O indicated that these low values were not solely due to differences in circulation. Decreased uptake was not accompanied by equivalent decreases in the concentration of aspartate and glutamate in the tumors. There were small changes in the net uptake of these amino acids in the slowly growing hepatoma 7787 and no significant differences in regenerating liver and hepatoma 5123C, a tumor of intermediate growth rate. The net uptake of [14C]arginine and [14C]lysine in the hepatomas was similar to that in host livers, except for a 250% increase in uptake of [14C]lysine in hepatoma 5123C. A decreased uptake of the magnitude seen with dicarboxylic amino acids in rapidly growing hepatomas has not been observed with other amino acids.     

In vivo imaging of brain incorporation of fatty acids and of 2-deoxy-D-glucose demonstrates functional and structural neuroplastic effects of chronic unilateral visual deprivation in rats

Regional cerebral 'incorporation coefficients' k* of each of 3 labeled long-chain fatty acids -[9,10-3H]palmitate ([3H]PA), [1-14C]arachidonate ([14C]AA) and [1-14C]docosahexaenoate ([14C]DHA)-were measured using quantitative autoradiography in 11 bilateral brain visual areas of 3.5-month-old awake, hooded, Long-Evans rats, and were compared with regional cerebral metabolic rates for glucose (rCMRglc). The rats, which had undergone unilateral orbital enucleation at 15 days of age, were studied either in the dark with eyelids of the intact eye sutured, or when stimulated in a light box with the intact eye open. rCMRglc did not differ between homologous contralateral and ipsilateral visual areas in the dark or during stimulation, but was elevated bilaterally by 25% or more in many visual areas during stimulation compared with dark. Contralateral compared with ipsilateral k* was lower for each fatty acid tracer in superficial gray of the superior colliculus (in dark and during stimulation) and dorsal nucleus of lateral geniculate body (during stimulation). In the dark, k* for [3H]PA was correlated significantly with rCMRglc for the 22 visual areas studied, whereas during stimulation k* for [14C]AA was correlated with rCMRglc. These results suggest that central neuroplastic changes following chronic unilateral enucleation are accompanied by reduced incorporation of [3H]PA, [14C]AA and [14C]DHA into contralateral brain ares that normally receive crossed retinofugal fibers, and by symmetry of rCMRglc in the dark but increased bilateral symmetrical responsiveness of rCMRglc to visual stimulation of the intact eye.     

P53 genes and malignant hematologic diseases

The primary objective of the present study was to compare the rates of plasma clearance and hepatic utilization of stearic (18:0), myristic (14:0) and linoleic (18:2) acids, as introduced via chylomicrons. Lymph chylomicrons were specifically labeled in vivo with [14C]stearic and (SA), [14C]myristic acid (MA), or [14C]linoleic acid (LA) by infusing donor rats intraduodenally with the labeled fatty acids in a lipid emulsion. Following intravenous injection of recipient rats with the labeled chylomicrons, the rates of plasma clearance and incorporation of the label in triglycerides (TG), phospholipids (PL) and other lipids in the liver were compared at 5, 15 and 30 min. [14C]SA was cleared at a slightly faster rate (t1/2 = 7.0 min) than [14C]MA (t1/2 = 8.1 min) and [14C]LA (t1/2 = 8.0 min) (P < 0.05). [14C]SA was accumulated in the liver at a significantly faster rate than [14C]MA and [14C]LA. At the peak (15 min) of hepatic uptake, 30.3% of [14C]SA, 26.2% of [14C]LA and 21.9% of [14C]MA were recovered in the liver. At 30 min, 33.5% of [14C]SA was taken up by the liver, whereas 27.8% of [14]LA and only 15.2% of [14C]MA were removed. In the liver, the percentage of [14C]SA incorporated into PL steadily increased with time, whereas the percent-age incorporated into TG decreased. [14C]SA was preferentially incorporated into PL at all time intervals, as compared with [14C]MA and [14C]LA. At 30 min, 38.6% of [14C]SA was found in PL, and only 5.2% of [14C]MA and 12.0% of [14C]LA were present in PL.(ABSTRACT TRUNCATED AT 250 WORDS)     

8-[18F]fluorooctanoic acid and its beta-substituted derivatives as potential agents for cerebral fatty acid studies: synthesis and biodistribution

Fluorine-18 labeled analogs of 8-fluorooctanoic acid and its structurally modified derivatives with methyl or gem-dimethyl branching or with oxygen substitution at the C3 position were prepared using nucleophilic substitution of the tosylate precursors by [18F]fluoride ion, for evaluation as tracers for cerebral fatty acid metabolism. Tissue distribution studies in rats showed low brain uptakes of these 18F-labeled fatty acid analogs with poor brain-to-blood ratios of activity. The oxygen-substituted analog did not show any significant accumulation of radioactivity in most tissues. The initial brain uptake of activity after injection of ethyl 8-[18F]fluorooctanoate and its free acid remained virtually unchanged over an extended time period, beta-Monomethyl and beta-gem-dimethyl branched analogs had similar brain uptake at the early time period, but they showed rapid clearance of activity from the brain. TLC analysis showed no incorporation of 8-[18F]fluorooctanoic acid and its beta-dimethyl analogs into brain lipids. It was also shown in the metabolite analysis that the labeled metabolites produced from 8-[18F]fluorooctanoic acid are found in blood, and that they could enter the brain to a significant degree. On the contrary, such radioactive metabolites could not be found in the brain in the experiment with the beta-gem-dimethyl branched analog. Thus, the present studies showed that retention of radioactivity in the brain with 8-[18F]fluorooctanoic acid derivatives is mainly attributable to their radioactive metabolites, and that the rapid clearance of beta-branched analogs from the brain is due to the lack of availability as substrates in the cerebral fatty acid metabolism.     

Resistance of A-431 epidermoid carcinoma cells to early membrane changes induced by tumour promoters

The A-431 human epidermoid carcinoma cell line was resistant to 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibition of epidermal growth factor binding, enhanced incorporation of [3H]choline into phospholipids and uptake of 86Rb an [3H]2-deoxyglucose. The cells were also resistant to TPA-stimulated release of radioactive choline derivatives and arachidonic acid from cells prelabelled with [3H]choline or [14C]arachidonic acid, respectively. The A-431 cells did not metabolise [3H]TPA. Despite their TPA-unresponsiveness, A-431 cells contained specific, high affinity binding sites for [3H]phorbol-12,13-dibutyrate with characteristics similar to other cultured cell lines.     

Raman detection of macular carotenoid pigments in intact human retina

We are developing procedures to repeatedly and noninvasively image the expression of transplanted reporter genes in living animals and in patients, using PET. We have investigated the use of the Herpes Simplex Virus type 1 thymidine kinase gene (HSV1-tk) as a reporter gene and [8-14C]-ganciclovir as a reporter probe. HSV1-tk, when expressed, leads to phosphorylation of [8-14C]-ganciclovir. As a result, specific accumulation of phosphorylated [8-14C]-ganciclovir should occur almost exclusively in tissues expressing the HSV1-tk gene. METHODS: An adenoviral vector was constructed carrying the HSV1-tk gene along with a control vector. C6 rat glioma cells were infected with either viral vector and uptake of [8-3H]-ganciclovir was determined. In addition, 12 mice were injected with varying levels of either viral vector. Adenovirus administration in mice leads primarily to liver infection. Forty-eight hours later the mice were injected with [8-14C]-ganciclovir, and 1 hr later the mice were sacrificed and biodistribution studies performed. Digital whole-body autoradiography also was performed on separate animals. HSV1-tk expression was assayed, using both normalized HSV1-tk mRNA levels and relative HSV1-TK enzyme levels, in both the cell culture and murine studies. RESULTS: Cell culture, murine tissue biodistribution and murine in vivo digital whole-body autoradiography all demonstrate the feasibility of HSV1-tk as a reporter gene and [8-14C]-ganciclovir as an imaging reporter probe. A good correlation (r2 = 0.86) between the [8-14C]-ganciclovir percent injected dose per gram tissue from HSV1-tk positive tissues and HSV1-TK enzyme levels in vivo was found. An initial study in mice with [8-18F]-fluoroganciclovir and microPET imaging supports further investigation of [8-18F]-fluoroganciclovir as a PET reporter probe for imaging HSV1-tk gene expression. CONCLUSION: These results demonstrate the feasibility of using [8-14C]-ganciclovir as a reporter probe for the HSV1-tk reporter gene, using an in vivo adenoviral mediated gene delivery system in a murine model. The results form the foundation for further investigation of [8-18F]-fluoroganciclovir for noninvasive and repeated imaging of gene expression with PET.     

Fluoxetine and tricyclic antidepressants: clinical tolerance in short-term combined administration

No-carrier-added racemic [11C]metaraminol was prepared by a selective condensation of [11C]nitroethane with 3-hydroxy-benzaldehyde using tetrabutylammonium fluoride in tetrahydrofuran (THF) as a catalyst, followed by a reduction with Raney nickel in formic acid. [11C]Metaraminol was produced in 30 to 45% decay-corrected yield from [11C]nitroethane (13 to 20% decay corrected from [11C]CO2) within 45 to 55 min total synthesis time. Reversed phase high-performance liquid chromatography (HPLC) was used for the separation of the racemic erythro- and threo-forms of [11C]metaraminol. The radiochemical purity was higher than 98%, and the specific radioactivity at the end of synthesis was 500 to 800 Ci/mmol (18 to 30 GBq/mumol). Positron emission tomography (PET) examination of racemic erythro-[11C]metaraminol in a Cynomolgus monkey showed a high uptake of radioactivity in the heart. Following pretreatment with the selective norepinephrine reuptake inhibitor desipramine, the radioactivity uptake in the myocardium was markedly reduced (80%), demonstrating the specificity of erythro-[11C]metaraminol for the norepinephrine reuptake system of the heart. Pretreatment with desipramine had no effect on radioactivity in lung. The metabolism was rapid for [11C]metaraminol. The amounts of the total radioactivity representing [11C]metaraminol in plasma, determined by HPLC, were 14% at 6 min and 8% at 34 min. The high specific uptake of racemic erythro-[11C]metaraminol indicates that enantiomerically pure (R,S)-[11C]metaraminol has potential for detailed mapping of the sympathetic innervation of the human myocardium.     

PET findings in a brain abscess associated with a silent atrial septal defect

Brain abscesses are classical complications of congenital heart disease (CHD) in children and adolescents. This association is rarely observed in adults. We report a 46-year-old man presenting a fronto-parietal abscess associated with an asymptomatic atrial septal defect. Positron emission tomography (PET) study revealed high uptake of L-[methyl-11C]methionine ([11C]methionine) and 2-[18F]fluoro-2-deoxy-D-glucose (FDG) around the brain abscess. We suggest (1) to exclude a silent cardiac malformation in the presence of a cerebral abscess of unknown source occurring in adults; (2) to consider the diagnosis of brain abscess in cases of high uptake of [11C]methionine and FDG in relation to a brain lesion.     

Cell density-dependent mitogenic effect and -independent cellular handling of epidermal growth factor in primary cultured rat hepatocytes

Brain swelling is a serious complication associated with focal ischemia in stroke and severe head injury. Experimentally, reperfusion following focal cerebral ischemia exacerbates the level of brain swelling. In this study, the permeability of the blood-brain barrier has been investigated as a possible cause of reperfusion-related acute brain swelling. Blood-brain barrier disruption was investigated using Evans Blue dye and [14C]aminoisobutyric acid autoradiography in a rodent model of reversible middle cerebral artery (MCA) occlusion. Acute brain swelling and cerebral blood flow (CBF) during ischemia and reperfusion were analyzed from double-label CBF autoradiograms after application of the potent vasoconstrictor peptide endothelin-1 to the MCA. Ischemia was apparent within ipsilateral MCA territory, 5 min after endothelin-1 application to the exposed artery. Reperfusion, examined at 30 min and 1, 2, and 4 h, was gradual but incomplete within this time frame in the core of middle cerebral artery territory and associated with significant brain swelling. Ipsilateral hemispheric swelling increased over time to a maximum (>5%) at 1-2 h after endothelin-1 but was not associated with a significant increase in the ipsilateral transfer constant for [14C]aminoisobutyric acid over this time frame. These results indicate that endothelin-1 induced focal cerebral ischemia is associated with an acute but reversible hemispheric swelling during the early phase of reperfusion which is not associated with a disruption of the blood-brain barrier.     

Conversion of D-arabinose to D-erythroascorbic acid and oxalic acid in Sclerotinia sclerotiorum

D-glycero-Pent-2-enono-1,4-lactone (trivial name: D-erythroascorbic acid) occurs in the phytopathogen, Sclerotinia sclerotiorum (Lib.) de Bary, where it has a potential role as precursor of oxalic acid. On Glc/yeast/malt medium, S. sclerotiorum produces only nominal amounts of D-erythroascorbic acid but even partial replacement of Glc by D-Ara increases production of erythroascorbic acid and oxalic acid. Use of D-[1-14C]-, -[3-14C]-, or -[6-14C]Glc and D-[5-3H]-, -[2-14C,5-3H]-, or -[UL-14C]Ara provide additional information on erythroascorbic acid biosynthesis and cleavage. The latter process resembles that obtained by peroxygenation of erythroascorbic acid in alkaline solution. An unknown erythroascorbic acid-like compound also occurs in both Glc- and Ara-based cultures.     

Kinetics of [14C]cholic acid in fulminant hepatic failure: a prognostic test

The kinetics of intravenously injected [14C]cholic acid have been investigated in 14 patients with fulminant hepatic failure, 24 to 36 hr after the development of grade IV encephalopathy. Radioactivity was measured in plasma samples and in the individual plasma bile acid fractions after separation by thin layer chromatography. Plasma disappearance curves of the free [14C]cholic acid were calculated by an iterative nonlinear least squares fitting procedure using a computer. The disappearance of total plasma radioactivity was similar in all patients and greatly prolonged compared with healthy subjects. However, the plasma disappearance of free [14C]cholic acid was significantly faster in the 8 patients who recovered consciousness than in the 6 who did not. Plasma disappearance of free [14C]cholic acid correlated highly significantly with the proportion of conjugated [14C]cholate in plasma. All patients in whom more than 70% of plasma radioactivity was in the conjugated fraction 3 hr after injection survived and left hospital, whereas all of those in whom less than 55% was conjugated died. Measuring the percentage conjugation of [14C]cholate 3 hr after injection may therefore be a useful test of residual liver function in hepatic failure, as a guide to prognosis and in evaluating new forms of treatment.