Tumor uptake studies of S-adenosyl-l-[methyl-11C]methionine and l-[methyl-11C]methionine

Tumor accumulation of S-adenosyl-l-[methyl-¹¹C]methionine ([¹¹C]SAM) was investigated in mice bearing mammary carcinoma (FM3A) and in rats bearing ascitic hepatoma (AH109A). After injection of [¹¹C]SAM the blood clearance of ¹¹C radioactivity was rapid. The ¹¹C level was relatively high in both tumors. The uptake ratios of tumor to organ increased with time in several organs, especially in brain and muscle. In FM3A tumor tissue the ¹¹C was incorporated with time into the acid-precipitable fraction and 38% of the ¹¹C was detected in this fraction at 60 min after injection. This fraction reflects the amount of ¹¹C-methyl group transferred into macromolecules in tumor tissue. In AH109A-bearing rats the metabolisms of [¹¹C]SAM and l-[methyl-¹¹C]methionine ([¹¹C]Met), in vivo precursor of SAM, were compared. Tumor uptake of [¹¹C]SAM was about two thirds of that of [¹¹C]Met at 20 min after injection. At this time, for the [¹¹C]SAM 27 and 8% of the ¹¹C in the AH109A tissue were detected in the acid-precipitable and the lipid fractions, respectively. The corresponding figures for [¹¹C]Met were 61% and 2%. In the liver considerable amounts of ¹¹C were observed in the lipid fraction for both tracers.These results show that [¹¹C]SAM has potential as a tracer for tumor localization with positron emission tomography (PET) and suggest that in tumor studies combining [¹¹C]Met and PET, it should be taken into account that the ¹¹C-labeled methyl group of [¹¹C]Met is not only incorporated into protein but also other macromolecules and lipids via [¹¹C]SAM.     

Tumor diagnosis by pet: potential of seven tracers examined in five experimental tumors including an artificial metastasis model

The potential of seven tracers for the metabolic imaging of tumors by positron emission tomography was studied using five experimental tumor models. The tracers examined were 2-deoxy-2-[¹⁸F]fluoro-d-glucose ([¹⁸F]FDG), 2-deoxy-2-[¹⁸F]fluoro-d-galactose (2-[¹⁸F]FdGal) and 2-deoxy-2-[¹⁸F]fluoro-l-fucose (2-[¹⁸F]FdFuc) for investigating energy metabolism. l-[methyl-¹¹C]Methionine ([¹¹C]Met) and 6-[¹⁸F]fluoro-l-fucose (6-[¹⁸F]FFuc) were used for assessing protein and glycoprotein synthesis, while [³H]thymidine ([³ H]Thd) and 2-deoxy-5′-[¹⁸F]fluorouridine ([¹⁸F]FdUrd) were used to investigate nucleic acid metabolism. The highest mean uptake by the five different tumors was found for [³H]Thd, followed in order by [¹⁸F]FDG, [¹¹C]Met, 2-[¹⁸F]FdGal, [¹⁸F]FdUrd, 2-[¹⁸F]FdFuc and 6-[¹⁸F]FFuc. The tumor-to-tissue uptake ratios indicated that the nucleosides, [¹¹C]Met and 6-[¹⁸F]FFuc were better tracers in the brain region. All the tracers except for the fucose analogs were suitable for the thoracic region, while [¹¹C]Thd and [¹⁸ F]FDG were superior in the abdominal region. In comparison with the primary tumor model of Lewis lung carcinoma (3LL), [³H]Thd uptake in the artificial metastatic 3LL model showed the maximum enhancement, followed by [¹⁸F]FDG, [¹¹C]Met and the other tracers. The [¹⁸F]FDG uptake correlated with the [³H]Thd uptake. [¹⁸F]FdUrd, 6-[¹⁸F]FFuc and 2-[¹⁸F]FdGal could be used for distinguishing different types of tumors. The combined use of these radiotracers can possibly allow the assessment of tumor metabolism, and this indicates the viability of tumors.     

Comparison of synaptosomal and glial uptake of pipecolic acid and GABA in rat brain

The active uptake of [³H]pipecolic acid increased with incubation time and its uptake at 3 min was half of that at 20 min. [¹⁴C]GABA uptake rose earlier, and its uptake at 3 min was almost 80% of that at 20 min. On the other hand, a ratio (pellet/medium) of [³H]pipecolic acid uptake into glial cell-enriched fractions, was much less (0.4–0.6) than that of [¹⁴C]GABA (25.8–74.1). GABA, 10^–4 M, and pipecolic acid, 10^–4 M, produced a significant inhibition of [³H]pipecolic acid uptake into P₂ fractions. Pipecolic acid, 10^–4 M, significantly reduced the synaptosomal and glial uptake of [¹⁴C]GABA. GABA, 10^–4 M, affected neither spontaneous nor high K⁺-induced release of [³H]pipecolic acid from brain slices. It is suggested that pipecolic acid is involved in either synaptic transmission or in its modulation at GABA synapses in the central nervous system.     

Uptake of 2-fluoro-2-deoxy-d-[U-14C]-glucose during chemotherapy in murine Lewis lung tumor

Mice bearing intramuscular Lewis lung tumor were treated with BCNU and doxorubicin (ADM) to study chemotherapy-induced changes in the uptake of 2-fluoro-2-deoxy-[U-¹⁴C]glucose (FDG). A decreased FDG uptake, tumor regression and a diminished proportion of aneuploid versus diploid cells as evaluated by DNA flow cytometry were seen after treatment with BCNU but not with ADM; HPLC indicated that most of the ¹⁴C activity in tumors was from FDG6-phosphate. The results suggest that changes in FDG uptake reflect the effectiveness of antitumor therapy. FDG may be valuable in follow-up studies of cancer treatment.     

Investigation of tumor metastatic potential with N-[18F]fluoroacetyl-d-glucosamine

In order to investigate the metastatic potential of tumors in vivo by measuring hyaluronic acid metabolism, C57BL/6 mice with B16 melanoma variants and C3H/He mice with FM3A tumor variants were evaluated using N-[¹⁸F]fluoroacetyl-d-glucosamine (¹⁸F-GlcNFAc). The uptake of ¹⁸F-GlcNFAc was slightly higher (P < 0.05) in B16-F10 tumors (high metastatic potential) than in B16-F1 (low metastatic potential). Analysis of metabolites showed that acid-insoluble fraction was the largest one in the liver by 60 min, whereas in the tumors, phosphates fraction was the major metabolite. Slower metabolism in tumors was suggested, and it may be one of the reasons for the difficulty of detecting the characteristics of their hyaluronic acid synthesis. ¹⁸F-GlcNFAc uptake by FM3A variants showed no significant correlation with their metastatic potential. In addition, N-acetyl-d-[l-¹⁴C]glucosamine, 2-deoxy-d-[l-¹⁴C]glucose and [6-³H]thymidine failed to demonstrate any difference between tumors' metastatic variants in vivo.     

Vitamin K dependent formation of gamma-carboxyglutamate residues in tumor microsomes

Vitamin K stimulated the incorporation of ¹⁴C into proteins when microsomes from melanoma, mammary gland, mast cell and lymphoma tumors were incubated with Na₂¹⁴CO₃. The ¹⁴C label in the [¹⁴C] proteins was identified as [¹⁴C] γ-carboxyglutamate (Gla), which is formed by carboxylation of glutamic acid residues. Carboxylation in tumor microsomes ranged from 2 to 19% of the carboxylation in normal liver microsomes per mg of microsomal protein. Carboxylation in microsomes was completely blocked by 10 μM Warfarin. SDS-polyacrylamide gel analysis of the melanoma [¹⁴C] Gla protein(s) revealed one major peak of ¹⁴C with an apparent MW of less than 6,000.     

Synthesis and biodistribution of [11C]fludiazepam for imaging benzodiazepine receptors

As a tracer for in vivo studies on benzodiazepine receptors, 7-chloro-1,3-dihydro-5-(2-fluorophenyl)-1-[¹¹C]methyl-2H-1, 4-benzodiazepin-2-one, [¹¹C]fludiazepam, was synthesized by the methylation of norderivative with [¹¹C]CH₃I, and purified by high-performance liquid chromatography. Within 60 min [¹¹C]fludiazepam was obtained for injection in high radiochemical yields and in high radiochemical purity with a specific activity of up to 230mCi/μmol.After i.v. injection of [¹¹C]fludiazepam in rats the radioactivity was rapidly incorporated into many tissues and the blood clearance of the radioactivity was very rapid. The brain uptake was high and decreased gradually. The adrenal uptake was the highest and decreased with high loading doses. The effect of the loading dose on the uptake was also found in the heart and lungs. By autoradiography using [¹¹C]fludiazepam, a higher accumulation was visualized in the cortex and thalamus than in other regions.     

Peptide transport in yeast: uptake of radioactive trimethionine in Saccharomyces cerevisiae.

The synthesis of Met-Met-[¹⁴C]Met and [³H]Ac-Met-Met-Met are reported. Uptake of these compounds by Saccharomyces cerevisiae is examined in detail. The tripeptide Met-Met-Met is taken up by S. cerevisiae by a saturable system that is pH and temperature dependent and exhibits an apparent K_m of 7.7 × 10^?5m. Neither methionine nor dimethionine effectively compete with (Met)₃ uptake. The tripeptides Met-Ala-Met, Met-Met-Leu, (Ala)₃, (Leu)₃ and l-Met-l-Met-l-Met-OMe compete with (Met)₃ uptake whereas Ac(Met)₃, d-Met-d-Met-d-Met-OMe, (Lys)₃, and Gly-Met-Gly do not. Metabolic inhibitors (sodium azide, dinitrophenol, and potassium cyanide) completely prevent (Met)₃ uptake. Trimethionine is rapidly hydrolyzed by cellular peptidases after entry into the cell. Ac(Met)₃ is transported by a system that is pH and temperature dependent with an apparent K_m of 16 × 10^?5m.     

Tumor uptake studies of d,l-[5-14C]ornithine and d,l-2-difluoromethyl[5-14C]ornithine

The feasibility of d,l-[5-¹⁴C]ornithine ([¹⁴C]ornithine), a precursor for polyamine synthesis, and d,l-2-difluoromethyl[5-¹⁴C]ornithine ([¹⁴C]DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC) were investigated for tumor localization. As an animal model, mice bearing mammary carcinoma, FM3A, were used. After i.v. injection of [¹⁴C]ornithine accumulation of radioactivity was observed in the FM3A, in which 43% of the ¹⁴C radioactivity was measured in the polyamine pool and 41% in the amino acid pool at 60 min after injection. Tumor uptake of [¹⁴C]DFMO was relatively low but constant during 60 min after injection. At 60 min after injection, 11% of the ¹⁴C was present in the acid-precipitable fraction of the FM3A, which suggests the formation of an irreversible complex of [¹⁴C]DFMO with ODC. For both compounds rapid blood clearance and high tumor-to-organ ratios were observed. Our results indicate that in connection with an enhanced polyamine synthesis in the tumors, the compounds investigated have potential as tracers for tumor detection.     

Control of fatty acid incorporation in membrane phospholipids. X-ray-induced changes in fatty acid uptake by tumor cells

Lymphosarcoma cells isolated from the spleens of tumor-bearing mice were used to study the effect of a low dose of X-rays (5 Gy) on the incorporation of [³H]palmitate and [¹⁴C]arachidonate into the lipids of the tumor cells. Palmitate and arachidonate were rapidly incorporated especially into the phospholipids of the cells. Between one and three hours after the start of the incubation with radiactive palmitate 80–90% of the label of the total lipids was found in the phospholipid fraction. Already after a few minutes of incubation with radioactive arachidonate, about 95% of the label was incorporated in the phospholipids. Irradiation caused a small but significant increase in the rate of fatty acid incorporation for both fatty acids. Concomitantly, a significantly increased amount of fatty acid was removed from the medium by the cells as a result of the irradiation, and the specific radioactivity of the free fatty acids in the cells was found to be enhanced. The radiation effect on the tumor cells could be mimicked by a hypotonic treatment. The magnitude of the radiation-induced stimulation of the fatty acid incorporation was similar to that of the hypotonically induced effect. Cells which had received a hypotonic treatment before the irradiation, did not show an additional radiation-induced enhancement of fatty acid incorporation into the cellular lipids. When the cells were incubated with serum albumin loaded with a relatively large (non-physiological) amount of complexed fatty acids (fatty acid: albumin molar ratio, $\text{ν}\text{= 3.7}$), no radiation effect on the fatty acid incorporation could be detected. It is concluded that hypotonic treatment, irradiation, and increased supply of exogenous fatty acids all lead to an enhanced flux of fatty acids into the cells. These results confirm our previous suggestion that the uptake of fatty acids through the plasma membrane is the rate-limiting step in the fatty acid incorporation into the phospholipids and that ionizing radiation is one of the means to enhance fatty acid uptake through the plasma membrane leading to an increased incorporation into the phospholipids.     

The uptake of organic molecules by the ventral tube of Tomocerus flavescens (Tullberg, 1871) (Insecta: Collembola)

The net influx of water-soluble organic molecules by the ventral tube of Tomocerus flavescens, a soil litter-inhabiting Collembolan, was investigated. The following substances were tested: [¹⁴C]urea, [¹⁴C]glycerol, [¹⁴C]erythritol, [¹⁴C]l-leucine, [¹⁴C]d-glucose, [³H]inulin. The animals were exposed to moist filter paper containing a specific test solution. When they evert their ventral-tube vesicles, they absorb water and solutes through the cuticle and the transport epithelium into the body haemolymph. Contamination by radioactive substances and oral solute uptake was avoided by an experimental device. It is evident that the uptake rates decrease with increasing molecular mass especially in a range of 100–200. Further, the rates correlate with the radius of hydrated molecules and their lipid solubility. Significant differences in urea uptake have been shown for animals less than 10 days in culture (“field” animals) and more than 10 days in culture (“laboratory” animals). Whether changes in cuticle permeability could be affected by abrasion is discussed. There is a high deviation amongst uptake values in all experimental series. It seems probably that, besides individual differences caused by abrasion, the animals differ physiologically, e.g. during the moulting cycle and seasonally. A nutritive function of the ventral tube seems to be unlikely. Calculation reveals that the absorbed glucose provides only 0.013% of the amount the animals need for respiration.     

Metabolism of phenylalanine and tyrosine in tobacco cell lines resistant and sensitive to p-fluorophenylalanine

Chromatography of soluble polyphenols of p-fluorophenylalanine-sensitive and -resistant tobacco cells revealed that the 10-fold increased level found in the resistant line was largely due to the accumulation of two unidentified polyphenols. The uptake of Phe-[U-¹⁴C] and Tyr-[U-¹⁴C] by the resistant line was ca 10 % that by the sensitive line. About 90 % of the phenylalanine-[¹⁴C] which was taken up by both cell lines could be accounted for as free phenylalanine in protein, soluble polyphenols or CO₂. The fate of Tyr-[¹⁴C] was similar to that of phenylalanine except that the incorporation was into insoluble polymeric forms of polyphenols rather than into soluble polyphenols. The resistant line incorporated 9-times more phenylalanine-[¹⁴C] into soluble polyphenols than did the sensitive line. The different ¹⁴C-aromatic amino acid accumulation and incorporation patterns noted with the two cell lines indicates that there are different active pools. Differential uptake rates by the two cell lines might affect the distribution of the absorbed amino acid among the different pools.     

Noradrenaline, a Transmitter Candidate in the Retina

The occurrence, metabolism, uptake, and release of noradrenaline were studied in the bovine retina with the following results. (1) Small amounts of noradrenaline occur in the retina and are restricted to the area corresponding to the inner nuclear and plexiform layers. (2) Retinal tissue can metabolise [¹⁴C]dopamine to form quantities of [¹⁴C]noradrenaline. (3) [¹⁴C]Noradrenaline can also be partly metabolised to form [¹⁴C]normetanephrine. (4) When bovine retinas were incubated with 5 × 10^-7 M-[³H]noradrenaline for 20 min and processed for autoradiography, most of the label was associated with apparent nerve processes in the inner plexiform layer. Biochemical analysis showed that more than 95% of the label was noradrenaline. (5) [¹⁴C]Noradrenaline uptake saturated with increasing noradrenaline concentrations and followed Michaelis-Menten kinetics. This uptake could be accounted for by two processes, a high-affinity system with a K_m1 of 5 × 10^-8 M and a V_max1 of 0.193 pmol/mg/10 min and a low-affinity system with a K_m2 of 6.3 × 10^-5 M and a V_max2 of 0.109 nmol/mg/10 min. (6) Noradrenaline uptake was strongly dependent on temperature and sodium, less dependent on potassium, and independent of calcium and magnesium ions. (7) Centrally acting drugs, such as desipramine, imipramine, desmethylimipramine, and amitriptyline, inhibited noradrenaline uptake by more than 55% at the concentration of 5 × 10^-5 M. These drugs at the same concentration diminished dopamine uptake by less than 30%. (8) Noradrenaline uptake is stereospecific, the (-) isomer having a greater affinity for the uptake sites than the (+) isomer. (9) [¹⁴C]Noradrenaline in the retina could be released by increasing the external potassium concentration. This release was calcium-dependent and was blocked by 20 mM-cobalt chloride. The present studies could be interpreted as supporting the idea that noradrenaline acts as a transmitter in the retina.     

Contribution of Particle-Bound Bacteria to Total Microheterotrophic Activity in Five Ponds and Two Marshes

We examined the abundance and heterotrophic uptake of bacteria attached to particulate matter suspended in five coastal ponds and two marshes near Woods Hole, Mass. Although the number of particle-bound bacteria was low (<10%), these bacteria incorporated a large proportion (>40%) of [¹⁴C]glucose and [¹⁴C]glutamate in selected aquatic systems. The uptake per cell was significantly higher for epibacteria than for unattached bacteria in all systems. Two groups of the aquatic environments sampled were statistically different in the contribution made by particle-bound bacteria to total bacterial abundance and to total assimilation of [¹⁴C]glucose and [¹⁴C]glutamate. Particle-bound bacteria were more important in those waters with a high particle concentration and not flushed regularly by tides than in waters with a low particle concentration and flushed regularly.     

Targeting of auxin carriers to the plasma membrane: differential effects of brefeldin A on the traffic of auxin uptake and efflux carriers

Monensin and brefeldin A (BFA), inhibitors of Golgi-mediated protein secretion, rapidly perturb the transport catalytic activity of specific plasma membrane-associated efflux carriers for indole-3-acetic acid (IAA) and inhibit polar transport of IAA. To determine if these responses result solely from perturbation of the efflux carrier or whether specific auxin uptake carrier function is also affected, the influence of BFA on the cellular transport of a range of auxins with contrasting affinities for specific auxin uptake and efflux carriers was investigated in zucchini (Cucurbita pepo L.) hypocotyl tissue. In-flight addition of BFA (3 · 10⁻⁵ mol · dm⁻³) caused a rapid (lag < 10 min) and substantial (fourfold) increase in the rate of [1-¹⁴C]IAA net uptake by zucchini hypocotyl tissue. In the presence of the specific auxin efflux carrier inhibitor N-1-naphthylphthalamic acid (NPA; 3 · 10⁻⁶ mol · dm⁻³), BFA slightly reduced the rate of [1-¹⁴C]IAA net uptake. Stimulation of [1-¹⁴C]IAA net uptake by BFA was concentration-dependent. In the absence of BFA, net uptake of [1-¹⁴C]IAA exhibited the characteristic biphasic response to increasing concentrations of competing cold IAA but in the presence of BFA, [1-¹⁴C]IAA uptake decreased smoothly with increase in concentration of competing unlabelled IAA, indicating a loss of auxin efflux carrier activity but retention of functional uptake carriers. The half-time for mediated efflux of [1-¹⁴C]IAA from preloaded zucchini tissue was substantially increased by BFA (t_1/2 = 51 min, controls; 107 min, BFA-treated). Treatment with BFA and/or NPA did not significantly affect the net uptake by, or efflux from, zucchini tissue of [1-¹⁴C]2,4-dichlorophenoxyacetic acid ([1-¹⁴C]2,4-D), a substrate for the auxin uptake carrier but not the auxin efflux carrier. Uptake of [1-¹⁴C]2,4-D declined smoothly with increasing concentrations of competing unlabelled IAA whether or not BFA was included in the uptake medium, confirming the failure of BFA to perturb auxin uptake carrier function. Transport of 1-[4-³H]naphthaleneacetic acid (1-NAA) exhibited little response to BFA or NPA, confirming that it is only a weakly transported substrate for the efflux carrier in zucchini cells. Received: 12 November 1997 / Accepted: 27 January 1998     

Glucose handling by hepatocytes from obese Zucker rats

Hepatocytes isolated from obese Zucker rats showed a significantly higher rate of both [U-¹⁴C]glucose and [U-¹⁴C]lactate incorporation into [¹⁴C]lipid than those from their lean counterparts. This was associated with a marked increase in the lipogenic rate measured by the incorporation of³H₂O into the cell esterified fatty acids. Although there were no changes in the incorporation of the tracer into either [¹⁴C]glycogen or¹⁴CO₂, the [¹⁴C] total uptake was significantly higher in the obese animals. The high rate of [¹⁴C]lipid synthesis from glucose was observed both at 15 and 30 mM substrate concentrations and was linked to an enhanced uptake of the tracer into the cell as measured using the decarboxilation of [1-¹⁴C]glucose in the presence of phenazine methosulphate. The presence of insulin in the incubation medium had no effect on the uptake of glucose by the liver cells. However, the large uptake of glucose by the hepatocytes from the obese animals was not related to an enhanced rate of transport as measured using 3-O-methyl[U-¹⁴C]glucose. The activity of glucose-6-phosphate dehydrogenase together with a higher [1-¹⁴C]glucose/[U-¹⁴C]glucose descarboxylation ratio indicate a predominant very active pentose phosphate pathway which may be responsible for the enhanced glucose uptake observed in the hepatocytes from the obese animals.     

Experimental study of solute transport and extraction by a single root in soil

The fate of ¹⁴C-2,4,6-trinitrotoluene ([U-¹⁴C]TNT) in soil/plant systems was studied using onion (Allium cepa L.) plants with only a single root. It was found that the single roots grew exponentially and that the rate of water uptake of the onion plants increased exponentially, as well. The concentration of [U-¹⁴C] in the roots at first increased and then appeared to reach a steady state, while the [U-¹⁴C] concentration in the leaves was found to increase linearly with time. The [U-¹⁴C] concentration in the rhizosphere increased gradually, while in the bulk soil it decreased slowly. The accumulation of [U-¹⁴C] in the rhizosphere is likely to difference between movement into the rhizosphere (through advective mass flow of soil water by root uptake) and its uptake into the roots. The distribution of ¹⁴C in the soil/plant system was found to be 60–85% in the soil solid phase, 7–11% in the soil liquid phase, <1% in the soil air phase, <1% in the root compartment, and <0.01% in the leaf compartment. The maximum RCF (root concentration factor) value for TNT and its derivates was found to be about 20, and the maximum TSCF (transpiration stream concentration factor) was 0.18. These values can be changed by a variety of factors in soil-plant systems     

Studies of aluminum in rat brain

The effects of high aluminum concentrations in rat brain were studied using¹⁴C autoradiography to measure the uptake of [¹⁴C]2deoxy-d-glucose ([¹⁴C]2dG) and microbeam proteon-induced X-ray emission (microPIXE) with a 20-μm resolution to measure concentrations of magnesium, aluminum, potassium, and calcium. The aluminum was introduced intracisternally in the form of aluminum tartrate (Al-T), and control animals were given sodium tartrate (Na-T). The¹⁴C was administered intravenously. The animals receiving Al-T developed seizure disorders and had pathological changes, which included cerebral cortical atrophy. The results showed that there was a decreased uptake of [¹⁴C]2dG in cortical regions in which increased aluminum levels were measured, i.e., there was a correlation between the aluminum in the rat brain and decreased brain glucose metabolism. A minimum detection limit of about 16 ppm (mass fraction) or 3×10⁹ Al atoms was obtained for Al under the conditions employed.     

Thyroid hormone inhibition of synaptosome amino acid uptake and protein synthesis.

Abstract— 3,3′,5-Triiodothyronine (T₃) inhibited L-[¹⁴C]leucine uptake into synaptosomes. Inhibition was competitive with a K_i of 3.1 × 10^?5m . Hofstee plot revealed an inverted hyperbolic curve suggestive of a two carrier or carrier plus diffusion mediated system for amino acid uptake. Both the carrier mediated and diffusional components were inhibited by thyroid analogues. l -Thyroxine and analogues inhibited the incorporation of l -[¹⁴C] leucine into cerebral synaptosome protein. At 50 μm , the triiodo-compounds were more inhibitory than tetraiodo->3,5-triiodo-l -thyronine >3,3′,5-triiodothyropro-pionic> l -thyroxine >3,5-diiodo-l -tyrosine. Thyroid analogue inhibition was not seen in liver or brain mitochondrial protein synthesis. 3,3′,5-Triiodothyronine had no effect on respiratory control or 2,4-DNP stimulated synaptosome respiration supported by malate plus pyruvate. Ouabain did not inhibit [¹⁴C]leucine uptake into adult synaptosomes. There was synergistic inhibition of synaptosome protein synthesis by thyroid analogues in the presence of 0.2 mm -ouabain. 3,3′,5-Triiodothyronine had no effect on synaptosome fraction ATPase or Na-K ATPase. Addition of T₃ induced further inhibition of synaptosome protein synthesis in the presence of either chloramphenicol (100μm ) or cycloheximide (50μg/ml). [¹⁴C]Glycine uptake and incorporation into synaptosome protein was inhibited by 3,3′,5-triiodothyronine. There was no inhibition of [¹⁴C]proline uptake or incorporation. The above evidence and kinetic data strongly favor a selective competitive block in amino acid transport at the synaptosome membrane leading to a decreased rate of protein synthesis.     

Distinct patterns of entry of two non-metabolizable amino acids into brain and other organs of infant guinea pigs

Abstract— Entry of [3-¹⁴C] α-aminoisobutyric acid (AIB) and [1-¹⁴C] 1-aminocyclopentanecarboxylic acid (cycloleucine) into the brain and other organs of the infant guinea pig has been investigated in vivo. The entry of [¹⁴C]AIB into brain was markedly restricted in comparison to its entry into other organs. The mean distribution ratio (¹⁴C in tissue water/¹⁴C in plasma water) achieved in brain at 45 min after administration of a pulse of [¹⁴C]AIB was 0.3. All other organs studied concentrated [¹⁴C]AIB from the blood stream, with the greatest uptake occurring in liver and kidney, in which distribution ratios reached values of 5–10. In contrast to AIB, [¹⁴C]cycloleucine entered the brain at a rate approximately the same as that into other organs. Distribution ratios for [¹⁴C]cycloleucine ranged between 0.5 and 2.0 for all organs. During the first few days of postnatal life, there was a sharp increase of concentrative uptake of [¹⁴C]AIB into liver and kidney. The entry of [¹⁴C]AIB into brain remained unchanged during this period. There was a small (35 percent) decrease in the rate of entry of [¹⁴C]cycloleucine into brain during the first 3 days of postnatal life. Since [¹⁴C]AIB is known to be concentrated from the surrounding medium by brain slices in vitro, we concluded that the locus of restriction of the entry of [¹⁴C]AIB into the brain in vivo is at the blood-brain barrier. We hypothesize that this property of the barrier is important in preventing concentrative uptake of pharmacologically active and potentially harmful amino acids by brain tissue.