Respiratory chain inhibition by fullerene derivatives: hydrogen peroxide production caused by fullerene derivatives and a respiratory chain system

Fullerene is a new type of carbon allotrope. We have shown that the fullerene derivative C(60)-bis(N,N-dimethylpyrrolidinium iodide), a regio isomer mixture, inhibited Escherichia coli growth and dioxygen uptake caused by E. coli and glucose. This result indicates that the mechanism of the bacteriostatic effect is the inhibition of energy metabolism. In this study, we isolated two regio isomers of C(60)-bis(N,N-dimethylpyrrolidinium iodide) and studied their effect on E. coli growth and on respiratory chain activity. In dioxygen uptake caused by the inner-membrane and NADH, the effect of fullerene derivatives was biphasic. At low concentrations of both fullerene derivatives, dioxygen uptake was inhibited, whereas at high concentrations, it was increased. At high concentrations, consumed dioxygen was converted to H(2)O(2). An electrochemical study revealed that reduced fullerene derivatives react with dioxygen. This activity was closely related to a redox property of the isomers.     

Effects of lindane on the glucose metabolism in rat brain cortex cells.

The influence of 0.5 mM gamma-hexachlorocyclohexane (gamma-HCH, lindane) on glucose transport has been investigated using the analog 3-O-methyl-D-(U-14C)glucose. The glucose uptake was lineal for at least 10 sec. Preincubation of dissociated brain cortex cells with lindane decreased the transport of glucose with respect to the controls. The treatment of brain cortex cells with other organochlorine compounds indicated that the alpha-, delta-HCH isomers and dieldrin reproduced the same inhibitory pattern, while beta-HCH and endrin were inactive. The total radioactivity incorporated into CO2 from (U-14C) glucose in the cerebral cortex is also inhibited by lindane in a time dependent manner.     

Inhibitory effects of cis- and trans-resveratrol on noradrenaline and 5-hydroxytryptamine uptake and on monoamine oxidase activity

This study investigated for the first time the potential effects of cis- and trans-resveratrol (c-RESV and t-RESV) on noradrenaline (NA) and 5-hydroxytryptamine (5-HT) uptake by synaptosomes from rat brain, on 5-HT uptake by human platelets, and on monoamine oxidase (MAO) isoform activity. Both c-RESV and t-RESV (5-200 microM) concentration-dependently inhibited the uptake of [3H]NA and [3H]5-HT by synaptosomes from rat brain and the uptake of [3H]5-HT by human platelets. In both experimental models, t-RESV was slightly more efficient than c-RESV. Furthermore, in synaptosomes from rat brain, the RESV isomers were less selective against [3H]5-HT uptake than the reference drug fluoxetine (0.1-30 microM). On the other hand, both c-RESV and t-RESV (5-200 microM) concentration-dependently inhibited the enzymatic activity of commercial (human recombinant) MAO isoform (MAO-A and MAO-B) activity, c-RESV being slightly less effective than t-RESV. In addition, both RESV isomers were slight but significantly more selective against MAO-A than against MAO-B. Since the principal groups of drugs used in the treatment of depressive disorders are NA/5-HT uptake or MAO inhibitors, under the assumption that the RESV isomers exhibit a similar behaviour in humans in vivo, our results suggest that these natural polyphenols may be of value as structural templates for the design and development of new antidepressant drugs with two important biochemical activities combined in the same chemical structure: NA/5-HT uptake and MAO inhibitory activity.     

Species differences in the stereoselectivity of kappa opioid binding sites for [3H]U-69593 and [3H]ethylketocyclazocine.

Stereoselectivity of the binding sites for the specific kappa-opioid agonist [3H]U-69593, a benzeneacetamido based ligand was investigated in membrane suspension prepared from frog and rat brain, as well as guinea pig cerebellum, using the pure chiral forms of different unlabelled opiates. The ligand binding sites showed stereospecificity with at least three orders of magnitude differences in the affinities (measured as Ki values) of the opioid stereoisomer pairs both in rat and guinea pig membrane fractions. However, in frog brain membranes there was no substantial difference in potencies of the (-) and (+) isomers competing for the [3H]U-69593 binding sites. Another type of the kappa-site preferring opioid ligand, [3H]ethylketocyclazocine, a benzomorphan derivative was able to discriminate between (-) and (+) forms of the same compounds even in frog brain membrane preparation. Our data concerning binding profile of [3H]U-69593 in frog brain membranes are consistent with the observation that kappa opioid binding sites in frog (Rana esculenta) brain differ from those kappa-sites found in mammalian brains.     

The [(99m)Tc(N)(PNP)](2+) metal fragment: a technetium-nitrido synthon for use with biologically active molecules. The N-(2-methoxyphenyl)piperazyl-cysteine analogues as examples

The incorporation of a bioactive molecule into a nitrido-containing (99m)Tc-complex has been successfully achieved by using the [TcN(PNP)](2+) metal fragment. In this strategy, the strong electrophilic [TcN(PNP)](2+) metal fragment efficiently reacts with bifunctional chelating ligands having a pi-donor atom set, such as N-functionalized O,S-cysteine. The 2-methoxyphenylpiperazine (2-MPP) pharmacophore, which displays preferential affinity for 5HT(1A) receptors, was conjugated to the amino group of cysteine to obtain 2-MPPP-cys-OS, where 2-MPPP is 3-[4-(2-methoxyphenyl)piperazin-1-yl]propionate. The asymmetric Tc(V)-nitrido complexes, [(99g/99m)Tc(N)(PNP)(2-MPPP-cys-OS)] (PNP = PNP3, PNP4), were obtained in high yield (95%), by simultaneous addition of PNP and 2-MPPP-cys-OS ligand to a solution containing a starting (99g)/(99m)Tc-nitrido precursor. A mixture of syn and anti isomers was observed, the latter being the thermodynamically favored species. In vitro challenge experiments using the anti isomers with glutathione and cysteine indicated that no transchelation reaction occurs. Assessment of the in vitro 5HT(1A) receptor-affinity of the technetium complexes revealed that only the anti-PNP4 complex possesses some affinity for the receptor, but displayed negligible brain uptake in biodistribution studies in rats in vivo.     

Stereoselective effects of ketamine on dopamine,serotonin and noradrenaline release and uptake in rat brain slices

Ketamine (2-(2-chlorophenyl)-(1-methylamino)-cyclohexanone) is a rapid-acting dissociative general anaesthetic whose hallucinogenic properties have made it a popular drug of abuse. Ketamine comprises two optical isomers, with differing pharmacology. In the present study, the effects of (+)- and (-)-ketamine on stimulated efflux and reuptake of dopamine (DA), noradrenaline (NA) and serotonin (5-HT) were compared in isolated superfused slices of the rat caudatoputamen (CPu), ventral bed nucleus of the stria terminalis (BSTV) or dorsal raphe nucleus (DRN), respectively. Monoamine efflux was elicited by local electrical stimulation (20 pulses, 100 Hz trains) at tungsten microelectrodes and measured at adjacent carbon fibre microelectrodes using fast cyclic voltammetry (FCV). In CPu (+)-ketamine increased stimulated DA efflux and slowed DA reuptake in a concentration-dependent manner (25-200 microM). At 100 microM (+)-ketamine increased DA efflux by 109+/-20% (mean+/-S.E.M., n=13) of control values after 30 min (P<0.001 versus control) and prolonged uptake half-time (t(1/2)) by 76+/-38% (n=9, P<0.001) of control. In contrast (-)-ketamine (100 microM) had no effect on DA efflux or uptake. In DRN, both isomers (100 microM) increased stimulated 5-HT efflux. (-)-Ketamine had a larger effect (P<0.001), an 88+/-15% increase in 5-HT efflux (n=9) versus 46+/-10% (n=8) for the (+)-isomer. The isomers had similar effects on 5-HT uptake, increasing t(1/2) by approximately 200%. No evidence of stereospecificity was seen in BSTV: both isomers had small effects (+)- and (-)-ketamine (100 microM) increasing NA efflux by 43+/-10% (n=7, P<0.001) and 29+/-8% (n=7, P<0.001), respectively. The isomers also had identical effects on NA uptake, each increasing uptake t(1/2) by approximately 100%. In summary, our data show that the optical isomers of ketamine have strikingly different stereospecificity for the monoamine systems and one might predict, therefore, a different psychotomimetic potential.     

Acute exposure of L6 myotubes to cis-9, trans-11 and trans-10, cis-12 conjugated linoleic acid isomers stimulates glucose uptake by modulating Ca2+/calmodulin-dependent protein kinase II

Conjugated linoleic acid (CLA), a dietary fat, has been considered beneficial in metabolic syndrome. Despite several findings indicating that CLA improves glucose clearance, little information is available regarding the cellular dynamics of CLA on skeletal muscle. We sought to investigate the role of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in cis-9, trans-11(c9,t11) and trans-10, cis-12 (t10,c12) CLA isomer-mediated glucose transport by L6 myotubes. t10,c12-CLA stimulated both intracellular Ca(2+) release (Ca(i)(2+)) and CaMKII phosphorylation, whereas c9,t11-CLA showed only modest effects on both. Sequestering Ca(i)(2+) with BAPTA/AM abrogated the effect of both CLA isomers on Akt substrate-160 kDa (AS160) phosphorylation and glucose uptake by myotubes. Exposing myotubes to KN-93 or autocamtide 2-related inhibitory peptide to block CaMKII activity prevented both CLA isomers from inducing AS160 phosphorylation and glucose transport. Likewise, genetic knockdown of CaMKII in myotubes using siRNA completely abolished CLA isomer-mediated glucose uptake. These results indicate that CLA isomers require Ca(i)(2+)-CaMKII to mediate glucose uptake. Evidence that CaMKII blockers inhibit t10,c12-CLA-mediated AMP-activated protein kinase (AMPK) activation indicated that CaMKII acts upstream of AMPK in response to t10,c12-CLA. Lastly, CLA isomers stimulated the formation of reactive oxygen species but had no effect on stress-activated protein kinase/c-jun NH(2)-terminal kinase. These data establish that t10,c12-CLA acts via Ca(i)(2+)-CaMKII-AMPK-AS160 to stimulate skeletal muscle glucose transport, whereas the mechanism of c9,t11-CLA remains unclear. Given that impairments in muscle glucose utilisation are apparent in metabolic syndrome, delineating the molecular mechanisms by which CLA isomers mediate muscle glucose uptake may identify new approaches to manage this condition.     

Synthesis and biological evaluation of novel oxindole derivatives for imaging neurofibrillary tangles in Alzheimer's disease

This letter describes the synthesis and biological evaluation of a novel series of radioiodinated oxindole (OI) derivatives for detecting neurofibrillary tangles (NFTs) in the brains of patients with Alzheimer's disease (AD). In binding experiments in vitro, 2-oxindole (2-OI) and 3-oxindole (3-OI) derivatives showed affinity for tau aggregates. The 3-OI derivative 14 showed the highest affinity of these derivatives. In biodistribution experiments using normal mice, the OI derivatives displayed good uptake (2.4-2.5%ID/g at 2min) and clearance from the brain with time (0.6-1.4%ID/g at 30min). In fluorescence staining experiments using AD brain sections, 14 clearly stained NFTs. 3-OI may serve as a new molecular scaffold for developing novel NFT imaging agents.     

INHIBITION OF GABA UPTAKE IN RAT BRAIN SLICES BY NIPECOTIC ACID, VARIOUS ISOXAZOLES AND RELATED COMPOUNDS

—A variety of isoxazoles structurally related to muscimol (3-hydroxy-5-aminomethylisoxazole) were tested as inhibitors of the uptake of GABA and some other amino acids in rat brain slices, and of the activity of the GABA-metabolizing enzymes l -glutamate 1-carboxylyase and GABA:2-oxo-glutarate aminotransferase. A bicyclic derivative, 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol, proved to be a more potent inhibitor of GABA uptake than muscimol. Structure-activity studies on this derivative, which appeared to be a competitive inhibitor of GABA uptake, led to the findings that nipecotic acid (piperidine-3-carboxylic acid) is a powerful non-competitive inhibitor of GABA uptake, and that perhydro-1,2-oxazine-6-carboxylic acid is a relatively weak competitive inhibitor of GABA uptake.     

Inhibition of proliferation of HT-29 colon adenocarcinoma cells by carboxylate NSAIDs and their acyl glucuronides.

Many nonsteroidal anti-inflammatory drugs (NSAIDs) which have antiproliferative activity in colon cancer cells are carboxylate compounds forming acyl glucuronide metabolites. Acyl glucuronides are potentially reactive, able to hydrolyse, rearrange into isomers, and covalently modify proteins under physiological conditions. This study investigated whether the acyl glucuronides (and isomers) of the carboxylate NSAIDs diflunisal, zomepirac and diclofenac had antiproliferative activity on human adenocarcinoma HT-29 cells in culture. Included as controls were the carboxylate NSAIDs themselves, the non-carboxylate NSAID piroxicam, and the carboxylate non-NSAID valproate, as well as its acyl glucuronide and isomers. The compounds were incubated at 1-3000 microM with HT-29 cells for 24 hr, with [3H]-thymidine added for an additional 2 hr incubation. IC50 values were calculated from the concentration-inhibition response curves for thymidine uptake. The four NSAIDs inhibited thymidine uptake, with IC50 values about 200-500 microM. All of the NSAID acyl glucuronides (and isomers, tested in the case of diflunisal) showed antiproliferative activity broadly comparable to the parent drugs. This activity may stem from direct uptake of intact glucuronide/isomers followed by covalent modification of proteins critical in the cell replication process. However, hydrolysis during incubation and cellular uptake of liberated parent NSAID will play a role. In HT-29 cells incubated with zomepirac, covalently modified proteins in cytosol were detected by immunoblotting with a zomepirac antibody, suggesting that HT-29 cells do have the capacity to glucuronidate zomepirac. The anti-epileptic drug valproate had no effect on inhibition of thymidine uptake, though, surprisingly, its acyl glucuronide and isomers were active. The reasons for this are unclear at present.     

Anion exchange chromatographic separation of inositol phosphates and their quantification by gas chromatography

The direct measurement of mass of inositol trisphosphate from biologic samples is described. Separation of inositol monophosphate, bisphosphate, trisphosphate, and inositol tetrakisphosphate was achieved using anion exchange chromatography with a sodium sulfate gradient. In addition, separation of the isomers of each inositol phosphate was performed using HPLC procedures. The individual inositol phosphate fractions were subsequently dephosphorylated and desalted. The myo-inositol from each fraction was then derivatized to the hexatrimethylsilyl derivative and the myo-inositol derivatives were quantified by a novel gas chromatographic analysis using the hexatrimethylsilyl derivative of chiro-inositol as an internal concentration reference. This method is a reproducible and relatively rapid procedure for the direct quantification of inositol phosphate mass which overcomes many of the problems associated with the use of radiolabeled precursors. The method is a significant improvement over existing procedures for the quantitative determination of the mass of inositol phosphate by virtue of improved recovery, sensitivity, and technical simplicity. The applicability of this method is illustrated by the quantitative determination of inositol trisphosphate in response to norepinephrine stimulation of adult canine myocytes and cerebral cortical brain slices and by measurement of the isomers of inositol trisphosphate in isolated myocytes.     

INHIBITION OF THE UPTAKE OF GABA AND RELATED AMINO ACIDS IN RAT BRAIN SLICES BY THE OPTICAL ISOMERS OF NIPECOTIC ACID

R(-)-Nipecotic acid was a more potent inhibitor than the S(+)-isomer of the uptake of GABA, (+)-nipecotic acid, and β-alanine in rat brain slices. (-)-Nipecotic acid was an order of magnitude more potent as an inhibitor of GABA uptake than as an inhibitor of β-alanine uptake, whereas the (+)-isomer was less selective. (–)-Nipecotic acid was a weak inhibitor of L-proline uptake and of rat brain acetylcholinesterase activity. Kinetic studies showed that both isomers of nipecotic acid were competitive inhibitors of GABA uptake when added at the same time as GABA, but non-competitive inhibitors when preincubated with the tissue for 15 min before addition of GABA. The apparent slope inhibition constants, which were not influenced by preincubation, indicated that (–)-nipecotic acid has an affinity for the carrier some 5 times higher than that for (+)-nipecotic acid. (–)-Nipecotic acid stimulated the release of preloaded radioactive GABA from rat brain slices. These observations indicate that (–)-nipecotic acid is a substrate-competitive inhibitor of GABA which combines with the GABA carrier and is taken up. (?)-Nipecotic acid and (+)-2,4-diaminobutyric acid, on the basis of their absolute structures and inhibition kinetics, are proposed to interact in a similar way with the GABA transport system.     

The Synthesis and Activity of cis-and trans-2-(Aminomethyl) cyclopropanecarboxylic Acid as Conformationally Restricted Analogues of GABA

Abstract: The synthesis of cis -2-(aminomethyl) cyclopropanecarboxylic acid, a new analogue of GABA in a folded conformation, is described, as is also an improved preparation of trans -2-(aminomethyl) cyclopropanecarboxylic acid. When adminstered microelectrophoretically the trans isomer was more potent than GABA as a bicuculline-sensitive depressant of the firing of cat spinal neurons in vivo , whereas the cis-isomer was less potent than GABA and its effects appeared not to be sensitive to bicuculline methochloride. Trans -2-(aminomethyl) cyclopropanecarboxylic acid was a weak inhibitor of the sodium-dependent uptake of GABA by mini slices of rat cerebral cortex and a substrate for the GABA: 2-oxoglutarate aminotransferase activity in extracts of rat brain mitochondria. The cis isomer did not influence GABA uptake or aminotransferase activity and neither isomer reduced glutamate decar-boxylase activity in rat brain homogenates. Both cyclopropane isomers inhibited the sodium-independent binding of GABA to synaptic membranes from rat brain and their relative potencies together with those found for the stereochemically related unsaturated derivatives, cis -and trans -4-aminocrotonic acid, were broadly consistent with the activity observed for these compounds in vivo on cat spinal neurons. These studies reinforce the evidence that extended rather than folded conformations of GABA are active at most GABA recognition sites within the mammalian central nervous system.     

Tumour localization of uroporphyrin isomers I and III and their correlation to albumin and serum protein binding

We were the first to report the superiority of uroporphyrin I (UROP I) as a tumour localizer when compared to haematoporphyrin derivative (HPD). In this study, we compared both isomers of UROP, i.e. I and III, in a KHJJ mammary carcinoma mouse model. Six and 18 h after UROP administration, the tumour, skin and gut porphyrin (P) content was quantitated. Tumour UROP I levels were always at least 50% higher than UROP III in tumour, whereas both isomers were barely detectable in the skin and gastrointestinal tract. We then explored the possibility that tumour P uptake might relate in part to the affinity of circulating P to mouse serum proteins (MSP), in particular, the major binding protein constituent, albumin. Copro-P III, deutero-P 2,4 disulphonic acid (DP), proto-P IX (PP) and heptacarboxylic P I (Hepta I) which in our mouse tumour model do not localize in malignant tissue, were compared to UROP I and III. The P was mixed with 0.775 microM human serum albumin (HSA) at different molar ratios (HSA:P range 2-8) and the unbound P concentration quantitated using an Amicon CF-25 membrane cone with centrifugation. The percentage free P was significantly higher for UROP I (92-98%) than III (82-95%) and significantly more than that observed with non-tumour localizing P studied. Similar data were obtained with MSP. This is consistent with the notion that enhanced uptake and retention (particularly UROP I) by malignant neoplastic tissue might reflect a higher affinity for UROP by tumour constituents than by circulating proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of 4-Hydroxy-5-methyl-3(2H)-furanone,a Flavor Component in Shoyu (Soy Sauce)

An analytical method was developed for determining the four optical isomers of a new synthetic pyrethroidal insecticide, α-cyano-3-phenoxy-benzyl 2-(4-chlorophenyl)isovalerate (Fenvalerate, S–5602). The cyano group in S–5602 molecule was converted into l-menthyloxycarbonyl group by heating S–5602 with l-menthol and hydrochloric acid. The obtained diastereoisomer derivative (l-menthyl ester deriv.) was chromatographed on a μPorasil column (4 mm i.d. × 0.3 m) using a solvent system of hexane-ethyl acetate (500+7, v/v) as a mobile phase, and the four isomers of S–5602 were separated from one another within 20 min. The compositions of S–5602 isomers were determined from their peak areas.     

Preparation and biological characterization of isomeric 188Re(V) oxocomplexes with tetradentate S4 ligands derived from meso-dimercaptosuccinic acid for labeling of biomolecules

A new type of tetradentate S4 ligand has been synthesized by bridging two molecules of meso-2,3-dimercaptosuccinic acid for stable binding and easy conjugation of rhenium-188 to tumor targeting structures. The stereoisomeric tetrathiolato S4 ligands form very robust anionic five-coordinated oxorhenium(V) and oxotechnetium(V) complexes. Two routes for the preparation of the (188)Re(V) oxocomplexes with (iBu)2N(O)C-C(SH)C(SH)C(O)NH(CH2)3NH(CH2)3NHC(O)C(SH)C(SH)C(O)N(iBu)2 (ligand 1) and its hydrophilic crown ether derivative (ligand 2) were tested and optimized. Several isomers were separated by HPLC from the preparation solutions and characterized in vitro and in vivo. The identity of the species obtained was determined by comparison with the HPLC profiles of reference (185/187)Re analogues and (99/99m)Tc complexes which were characterized by ESI-MS. All of them were absolutely stable in rat and human plasma solutions. Challenge experiments with cysteine corroborated the high inertness of the isomers toward ligand exchange reactions. Various in vivo samples, taken off at different times from blood, intestine, and urine of rats, confirmed the high in vivo stability of the (188)Re-S4 complexes. Biodistribution studies using male Wistar rats were performed and exhibited a high uptake and fast clearance from the liver of the more lipophilic cis and trans isomers of complex I (log P(o/w) between 1.5 and 1.7), whereas the isomers of the hydrophilic complex II (log P(o/w) about -1.75) were rapidly excreted via the renal and the hepatobiliary pathway. The low level of activity in the stomach confirms good in vivo stability. Thus, these new (188)Re-S4 complexes fulfill the requirements for a stable and high specific activity labeling of biomolecules with rhenium-188.     

Stereospecificity of siderophore-mediated iron uptake in Rhodotorula pilimanae as probed by enantiorhodotorulic acid and isomers of chromic rhodotorulate

Rhodotorulic acid (RA), a dihydroxamate siderophore produced by Rhodotorula pilimanae, forms 3:2 complexes with ferric and chromic ions (M2RA3) at pH 7. Kinetically inert chromic complexes of RA have been separated into geometrical isomers and for the first time partially resolved into optical isomers. The three isomers delta-cis, delta-trans, and lambda-trans were characterized by their visible and circular dichroism spectra. Inhibition by both delta-isomers of radiolabeled ferric RA uptake in R. pilimanae was equally effective. However the lambda-cis isomer was significantly less effective as an inhibitor. Concentration-dependent uptake kinetics were performed with ferric RA and the ferric complex of synthetic enantio-RA, which form predominantly delta and lambda complexes, respectively. The lambda-enantio-Fe2RA3 was 50% less effective in supplying iron to R. pilimanae than was Fe2RA3. An additional synthetic analog of RA, which lacks a carbonyl group at the diketopiperazine ring, exhibited the same uptake rates as ferric RA. We conclude that stereoselective recognition of optical isomers takes place during iron uptake mediated by RA and that this recognition primarily involves the right-handed delta coordination "propellor" of the metal center and its adjacent functionalities.     

Differential Interaction of Phencyclidine-Like Drugs with the Dopamine Uptake Complex In Vivo

The phencyclidine (PCP) derivative, [3H]N-[1-(2-benzo[b]thiophenyl)cyclohexyl]piperidine ([3H]BTCP), was used to label in vivo the dopamine uptake complex in mouse brain. The striatum accumulated the highest level of total and specific binding. Drugs which bind to the dopamine uptake site inhibited [3H]BTCP binding on an order similar to their in vitro affinities for the high-affinity [3H]BTCP site. Drugs which label selectively other monoamine uptake complexes. PCP, or sigma recognition sites were ineffective at doses up to 40 mg/kg. PCP bound to and dissociated from the dopamine uptake complex very rapidly. N-[1-(2-Thienyl)cyclohexyl]pideridine (TCP) and (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) had no effect at any time or at any dose. These results imply that the pharmacological effects of PCP are due to its simultaneous interaction with the dopamine uptake complex and the PCP receptor. Conversely, TCP and MK-801, which have the same behavioral properties as PCP, exert their action only through the interaction with the PCP receptor.     

Postnatal hypoxic-ischemic brain injury alters mechanisms mediating neuronal glucose transport

We examined the effect of hypoxic ischemia and hypoxia vs. normoxia on postnatal murine brain substrate transporter concentrations and function. We detected a transient increase in the neuronal brain glucose transporter isoform (GLUT-3) in response to hypoxic ischemia after 4 h of reoxygenation. This increase was associated with no change in GLUT-1 (blood-brain barrier/glial isoform), monocarboxylate transporter isoforms 1 and 2, synapsin I (neuronal marker), or Bax (proapoptotic protein) but with a modest increase in Bcl-2 (antiapoptotic mitochondrial protein) protein concentrations. At 24 h of reoxygenation, the increase in GLUT-3 disappeared but was associated with a decline in Bcl-2 protein concentrations and the Bcl2:Bax ratio, an increase in caspase-3 enzyme activity (apoptotic effector enzyme), and extensive DNA fragmentation, which persisted later in time (48 h) only in the hippocampus. Hypoxia alone in the absence of ischemia was associated with a transient but modest increase in GLUT-3 and synapsin I protein concentrations, which did not cause significant apoptosis and/or necrosis. Assessment of glucose transporter function by 2-deoxyglucose (2-DG) uptake using two distinct techniques, namely positron emission tomography (PET) and the modified Sokoloff method, revealed a discrepancy due to glucose uptake by extracranial Harderian glands that masked the accurate detection of intracranial brain glucose uptake by PET scanning. The modified Sokoloff method assessing 2-DG uptake revealed that the transient increase in GLUT-3 was critical in protecting against a decline in brain glucose uptake. We conclude that hypoxic-ischemic brain injury is associated with transient compensatory changes targeted at protecting glucose delivery to fuel cellular energy metabolism, which then may delay the processes of apoptosis and cell necrosis.     

Synthesis, radiofluorination and pharmacological evaluation of a fluoromethyl spirocyclic PET tracer for central σ1 receptors and comparison with fluoroalkyl homologs

The spirocyclic σ(1) receptor ligand 1 (1'-benzyl-3-(fluoromethyl)-3H-spiro[[2]benzofuran-1,4'-piperidine]) was prepared in four steps starting from methoxy derivative 5. Due to its high σ(1) affinity (K(i)=0.74nM) and selectivity against several other relevant targets, 1 was investigated as (18)F-labeled PET tracer and its biological properties were compared with those of homologous fluoroalkyl derivatives 2-4. The fluoromethyl derivative 1 was faster metabolized in vitro than homologs 2-4. In contrast to the radiosynthesis of [(18)F]2-4, the nucleophilic substitution of the tosylate 15 using the K[(18)F]F-K(222)-carbonate complex required heating to 150°C in DMSO to achieve high labeling efficiencies. Whereas radiometabolites of [(18)F]2-4 were not detected in vivo in the brain of mice, two radiometabolites of [(18)F]1 were found. Analysis of ex vivo autoradiography images provided rather low target-to-nontarget ratio for [(18)F]1 compared with [(18)F]2-4. [(18)F]1 showed a fast uptake in the brain, which decreased continuously over time. The brain-to-plasma ratio of the radiotracer [(18)F]1 was only exceeded by the fluoroethyl tracer [(18)F]2.