Lymphokine-induced uptake of [14C]glucosamine, [14C]glucose,and [3H]leucine by macrophages

Lymphokine-activated (LK⁺) and control (LK^–) macrophages were cultured for 66 h and then pulsed with [¹⁴C]glucosamine. Uptake of [¹⁴C]glucosamine was greater in LK⁺ than in LK^– cultures. If, after 66 h, the medium was replaced with fresh medium and then pulsed with either [¹⁴C]glucose or [¹⁴C]glucosamine, the uptake of isotope was greatly reduced compared to cultures with no change of medium. However, uptake of both radiolabeled substances was still found to be greater in LK⁺ cultures than in LK^– cultures. Although uptake of both substances was enhanced by lymphokines, the uptake kinetics of each isotope was different. Under similar conditions the uptake of [³H]leucine was not enhanced by lymphokine activation. These data are interpreted to mean that LK⁺ macrophages are metabolically stimulated and utilize more glucose and glucosamine. The difference in kinetics implies a different utilization by macrophages for each substance.     

Autoradiography of [14C]paraquat or [14C]diquat in frogs and mice: accumulation in neuromelanin

The herbicide paraquat has been suggested as a causative agent for Parkinson's disease because of its structural similarity to a metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which may induce a parkinsonism-like condition. MPTP as well as its metabolite 1-methyl-4-phenylpyridine have melanin affinity, and the parkinsonism-inducing potency of MPTP is much stronger in species with melanin in the nerve cells. Autoradiography of [3H]MPTP in experimental animals has shown accumulation in melanin-containing tissues, including pigmented neurons. In the present whole body autoradiographic study accumulation and retention was seen in neuromelanin in frogs after i.p. injection of [14C]paraquat or [14C]diquat. By means of whole body autoradiography of [14C]diquat in mice (a species with no or very limited amounts of neuromelanin) a low, relatively uniformly distributed level of radioactivity was observed in brain tissue. Accumulation of toxic chemical compounds, such as paraquat, in neuromelanin may ultimately cause lesions in the pigmented nerve cells, leading to Parkinson's disease.     

Topography of basal glucose utilization in the hippocampus determined with [1-14C]glucose and [6-14C]glucose

The activity of the pentose phosphate shunt was assessed under basal conditions in subregions of the hippocampus by measuring the uptake and retention of [1-14C]glucose and [6-14C]glucose and their 14C-labelled metabolites. The relative and absolute retention of carbon-14 from each of the two compounds was nearly identical in all regions examined. For each compound, the highest accumulation of 14C occurred in the granule cell layer of the dentate gyrus and in the pyramidal cell layer. Relatively high retention of radioactivity was also found in the molecular layer of dentate gyrus and in the stratum lacunosum-molecular. The stratum radiatum and stratum oriens contained the lowest levels of radioactivity among hippocampal regions. The equal retention of radioactivity from [1-14C]glucose and [6-14C]glucose implies that pentose phosphate shunt activity is very low throughout the hippocampus under the conditions of this study. The uptake and retention of radioactivity was evaluated in different hippocampal regions 10 or 30 min following intravenous injection of [1-14C]glucose. Although there was significantly more radioactivity at 30 min than at 10 min, the same topographic pattern of radioactivity within the hippocampus was observed in rats after both survival periods, indicating that an equal fraction of the [1-14C]glucose utilized in different hippocampal regions is oxidized to 14CO2 under these conditions. Most regions of high glucose utilization in the hippocampus determined with [1-14C]glucose and [6-14C]glucose correspond to regions of intense histochemical staining for cytochrome oxidase reported in the literature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolic studies on the uptake of [14C]-histidine and [14C]-histamine and histamine synthesis by guinea-pig basophils, in vitro.

The uptake of [14C]-histidine and [14C]-histamine and the conversion of [14C]-histidine to [14C]-histamine was measured in suspensions of guinea-pig bone marrow cells rich in basophils. When comparable amounts of labelled histidine or histamine were added to equal numbers of basophils, the uptake of histidine was approximately forty-five times greater than that of histamine. Purified eosinophils, neutrophils and mononuclear cells incorporated only a small proportion of [14C]-histidine when compared to the basophil; [14C]-histamine uptake by all these cell types was virtually negligible. Histidine uptake and the amount of histamine formed de novo was directly related to the number of basophils, the time of incubation and the substrate concentration. Histidine uptake was decreased by agents which inhibit glycolysis, oxidative phosphorylation, Na + - K + -dependent ATPase, protein synthesis and RNA synthesis. Inhibition was demonstrable in a dose-dependent fashion and at concentrations which had no apparent effect on cell viability. Inhibitors of DNA synthesis, and of microtubule function, had no influence on histidine uptake. Cytochalasin B, an inhibitor of microfilament function, also decreased histidine uptake but only at concentrations previously showen to affect hexose transport. None of the agents tested affected the uptake of [14C]-histamine or the amounts of new histamine formed from the histidine that had been incorporated. These studies suggest that histidine is preferentially incorporated into the basophil; that the uptake depends on the integrity of a number of metabolic pathways, but that once the histidine is taken up these requirements do not apply to the formation of new histamine. In contrast, histamine appeared to diffuse passively, and in relatively small amounts, into all the cell types tested.     

Comparison of the rate of absorption and proteolysis of [14C]choleragen and [14C]bovine serum albumin in the rat jejunum.

[14C]choleragen was used to study the rate of disappearance of choleragen enterotoxin from the jejunum of rats. [14C]bovine serum albumin (BSA) was studied in a similar manner. Almost one-third of the labeled toxin had disappeared from the intestine after 6 h. Its rate of disappearance was the same in germfree rats as in conventional rats. The rate of proteolysis of [14C]choleragen and [14C]BSA by intestinal mucodal lysosomal enzymes was also studied. Neither was significantly degraded by neutral proteases; however, heat-inactivated toxin was. They were all degraded by acid proteases; however, the rate of BSA proteolysis was only one-third of that of toxin. Soybean trypsin inhibitor had no effect on the in vivo disappearance of toxin nor on the acid proteases. It did inhibit the neutral protease digestion of heat-treated toxin. Aprotinin and protamine inhibited disappearance in loops of gut but had no effect to inhibit degradation rates. Gangliosides inhibited both rates of disappearance and proolysis of toxin. These agents had some different effects on disappearance rates and proteolysis of BSA. The data indicate that cholera enterotoxin is absorbed by intestinal mucosal cells and is degraded by acid proteases in the cells.     

Metabolism of D-[3-3H]glucose, D-[5-3H]glucose, D-[U-14C]glucose, D-[1-14C]glucose and D-[6-14C]glucose in pancreatic islets in an animal model of type-2 diabetes

This study aims at exploring specific aspects of D-glucose metabolism, so far not yet investigated, in pancreatic islets from adult control rats and animals (STZ rats) injected with streptozotocin during the neonatal period. The latter animals, which represent a current model of type-2 diabetes, displayed a lower body weight, higher plasma D-glucose concentration and lower insulinogenic index than control rats. The protein, DNA and insulin content were all also lower in islets prepared from STZ, rather than control rats. In the presence of 10.0 mM D-glucose, the paired ratio between D-[U-14C]glucose oxidation and D-[5-3H]glucose utilization was also decreased in the islets from STZ rats. No significant difference between control and STZ rats was observed, however, in terms of the ratios between D-[3-3H]glucose and D-[5-3H]glucose utilization, between the generation of radioactive lactate from 14C-labelled D-glucose and tritiated D-glucose utilization and between D-[1-14C]glucose and D-[6-14C]glucose oxidation. These findings reinforce the view that the previously documented preferential impairment of the oxidative modality of glycolysis in islets from STZ rats contrasts with the absence of any major anomaly in other variables of D-glucose catabolism.     

Muscle capillary permeability for [14C]inulin and [51Cr]EDTA in human forearm

Capillary permeability of [14C]inulin and [51Cr]EDTA was examined in human forearm in five healthy, subjects by indicator diffusion technique. Injections of, initially [125I]albumin and [14C]inulin, and after 30 min resting, of [125I]albumin and [51Cr]EDTA, were given in a brachial artery. During light exercise of the forearm, blood was sampled in 2-s periods from a deep cubital vein primarily draining muscles. The plasma flow rate, calculated as the dose of [125I]albumin in the injectate divided by the area under the curve for the venous concentration of 125I, was, on average, 8.5 ml min-1 100 g-1 forearm. Assuming [125I]albumin is a partially permeable tracer, a correction for extraction of albumin was performed. This gave extraction fractions of 0.107 +/- 0.015 (mean +/- SEM) for [14C]inulin and 0.377 +/- 0.033 for [51Cr]EDTA, respectively. The capillary permeability surface area product per 100 g tissue (CDC) was for [14C]inulin 0.90 +/- 0.19, and for [51Cr]EDTA 3.31 +/- 0.38 ml min-1 100 g-1 forearm. The average of the ratios of the CDC values of [51Cr]EDTA to those of [14C]inulin, 4.0 +/- 0.5, is significantly higher than the corresponding ratio between the measured free diffusion coefficients in water at 37 degrees C, 3.07 +/- 0.002 (N = 36 and 17, respectively). This indicates that there is some degree of restriction for [14C]inulin (MW 5200) relative to [51Cr]EDTA (MW 340.2) and it points to an 'equivalent pore radius estimate' of about 160 A in human muscle capillaries.     

Foetal bile pigment handling after administration of [14C] haemin

1. Advanced techniques for intra-uterine surgery were used to study haem degradation in foetal sheep prepared in utero with indwelling jugular, carotid and biliary cannulas. [(14)C]haemin was administered I.V. to the foetus, and plasma disappearance, biliary excretion, placental transfer and tissue distribution of radioactivity were measured over a 5-8 hr period.2. 8-30% of the (14)C-label was recovered in foetal bile, about 40% of this as bilirubin and the rest as unidentified [(14)C]haemin derivatives. 4-21% was transferred across the placenta, appearing in maternal bile almost exclusively as [(14)C]bilirubin. Excretion of (14)C-label totalled 18-33%.3. Six adult sheep infused with [(14)C]haemin excreted 19-49% of the dose in the bile over 8 hr, one third as bilirubin.4. The amount of endogenous bilirubin excreted per unit time/unit wt. of foetal liver increased with increasing foetal wt.5. It is concluded that near-term foetal sheep have a maturing mechanism for haem catabolism. Haem is partially excreted in foetal bile as bilirubin. Another fraction is transferred across the placenta, probably after prior conversion to bilirubin. The remainder is converted to un-identifiable end-products. Total excretion is approximately as effective as that in adults.     

Pancreatic uptake of [2-(14)C]alloxan

The uptake of [2-(14)C]alloxan by the pancreatic gland was investigated in control and streptozotocin-induced diabetic (STZ) rats, using both in vitro and in vivo techniques. Whether after 10 to 60 min incubation of pieces of pancreas in the presence of [2-(14)C]alloxan or 60 min to 24 h after intravenous injection of [2-(14)C]alloxan to control and insulin-treated STZ rats, the radioactive content of the pancreas (dpm/mg wet weight) only represented, in the STZ rats, about two thirds of the reference value found in control animals. These findings indicate that insulin-producing islet B-cells participate to a sizeable extent to the overall uptake of [2-(14)C]- alloxan by the whole pancreatic gland, despite the fact that they account for no more than about one percent of the total pancreas mass. Hence, it should be possible to preferentially label the endocrine moiety of the pancreas, in the perspective of its imaging and quantification by a non-invasive procedure, by use of a suitable radiolabelled molecule selectively taken up by islet, as distinct from acinar, pancreatic cells.     

Measurement of local cerebral blood flow with iodo [14C] antipyrine

The autoradiographic diffusible tracer technique for the measurement of local cerebral blood flow was originally designed for use with the radioactive, inert gas 131I-labeled trifluoroiodomethane and is applicable only with tracers that exhibit unrestricted diffusion through the blood-brain barrier. Because of the technical problems associated with the use of gaseous tracers, a suitable nonvolatile tracer has been sought. [14C] Antipyrine has been used previously and found to be unsuitable because of limitations in its diffusion through the blood-brain barrier. An analogue of [14C]antipyrine, iodo [14C]antipyrine, exhibits higher partition coefficients than [14C]antipyrine between nonpolar solvents and water and might, therefore, be expected to diffuse more freely through the barrier. Its use as the tracer in the local blood flow technique leads to values considerably above those obtained with [14C]antipyrine in the rat and cat and essentially the same as those obtained with the gas trifluoro[131I]iodomethane in the cat. Iodo[14C]antipyrine appears, therefore, to be a satisfactory nonvolatile tracer for the measurement of local cerebral blood flow.     

High resolution autoradiographic determination of the topographic distribution of radioactivity in the hippocampal formation after injection of [1-14C]glucose or 2-deoxy[14C]glucose

Using high resolution autoradiography, the accumulation of radioactivity after intravenous injection of [1-14C]glucose was measured in the corpus callosum, hippocampus, dorsal hippocampal commissure, somatosensory cortex, inferior colliculus and pontine periaqueductal grey. Autoradiograms were prepared by thaw-mounting 4 micron frozen sections on nuclear emulsion-coated slides, and were evaluated quantitatively with a computer-assisted video system for automated counting of silver grains. In all brain regions examined, silver grain densities were greater in rats killed 30 min after injection of [1-14C]glucose compared to rats killed 10 min after injection. After intravenous injection of [1-14C]glucose or 2-deoxy[14C]glucose, the relative uptake and retention of radioactivity in different hippocampal subregions was compared. Striking differences were found in the hippocampus between 2-deoxy[14C]glucose and [1-14C]glucose autoradiograms. After injection of 2-deoxy[14C]glucose, there were large variations in the uptake and retention of radioactivity among different pyramidal cell fields. The CA 3 pyramidal cell field retained considerably more radioactivity than other pyramidal cell fields after injection of 2-deoxy[14C]glucose, while after injection of [1-14C]glucose, the retention of radioactivity was similar in all pyramidal cell fields. After [1-14C]glucose injection, the dentate gyrus contained relatively high levels of radioactivity and more 14C accumulated in the granular layer, compared to the molecular layer. In contrast, after 2-deoxy[14C]glucose injection, there was uniformly less radioactivity throughout the dentate gyrus when compared to rats injected with [1-14C]glucose and there was no preferential accumulation of 2-deoxy[14C]glucose in the granular layer compared to the molecular layer.(ABSTRACT TRUNCATED AT 250 WORDS)     

The uptake of [14C]glycine by slices of mammalian spinal cord

1. The accumulation of [(14)C]glycine by slices of mammalian spinal cord has been measured.2. When slices of rat cord were incubated at 37 degrees C in a medium containing [(14)C]glycine, tissue:medium ratios of about 30:1 were attained after a 40 min incubation.3. After incubations at 37 degrees C for 40 min, almost all (98%) the radioactivity in the tissue was present as unchanged [(14)C]glycine.4. The process responsible for [(14)C]glycine uptake showed many of the properties of an active transport system: it was temperature sensitive, required the presence of sodium ions in the external medium, was inhibited by dinitrophenol and ouabain and showed saturation kinetics.5. The estimated K(m) value of glycine was 3.1 x 10(-5)M, and V(max) was 0.48 mu-mole/min.g cord.6. The uptake of [(14)C]glycine was not affected by the presence of large molar excesses of L-histidine, L-proline, L-aspartate, L-glutamate, L-valine or GABA, but was inhibited in the presence of L-alanine and L-leucine.7. The uptake of [(14)C]glycine was not reduced by strychnine, but a significant reduction in uptake was produced by p-hydroxymercuribenzoate.8. The uptake of [(14)C]glycine by the grey matter of rabbit spinal cord was 2 to 6 times greater than the uptake by slices of white matter incubated under the same conditions.9. Rat cerebral cortex, cerebellar cortex and medulla also accumulated [(14)C]glycine, and the uptake by the tissue slices in vitro appeared to parallel the concentration of glycine in these areas in vivo.10. It is suggested that the glycine uptake system may represent a possible mechanism for the inactivation of glycine at inhibitory synapses in the spinal cord.     

Oxidation of [1,12-14C]dodecanedioic acid by rat pancreatic islets

Several aliphatic dioic acids were recently reported to stimulate insulin release in isolated rat pancreatic islets incubated at close-to-physiological D-glucose concentrations. In order to gain insight into the mode of action of these acids in pancreatic islet B-cells, the oxidation of [1,12-14C]dodecanedioic acid (5.0 mM) was now measured in rat islets. Expressed as pmol of [1, 12-14C]dodecanedioic acid equivalent, the production of 14CO2 was close to 1.0 pmol/islet per 120 min, representing about 8% of that attributable to the oxidation of D-[U-14C]-glucose (8.3 mM). The dioic acid and the hexose failed to exert any significant reciprocal effect upon their respective oxidation rate. These findings support the view that the insulinotropic action of dodecanedioic acid, and presumably other aliphatic dioic acids, is causally linked to their capacity to act as nutrients in pancreatic islet cells.     

Both [1-14C]glucose and 2-[1-14C]deoxyglucose produce selective iso-frequency labelling in the inferior colliculus of the cat with short stimulation periods

Both [1-14C]glucose and 2-[1-14C]deoxyglucose (2-DG) revealed selective autoradiographic labelling to tones in the inferior colliculus of the cat with short stimulation periods (5-15 min). With longer periods of stimulation (45 min), the selectivity disappeared with glucose but remained with 2-DG. At all stimulation intervals, 2-DG labelling was always more selective than that obtained with glucose. However, the selectivity seen with glucose was good enough to indicate that isotopes of glucose with short half-lives could still be employed to study human functional activity with the positron emission tomography technique, provided that short stimulation periods were used.     

Calibration of [14C]plastic standards for quantitative autoradiography of [125I]labeled ligands with Amersham Hyperfilm beta-max

The densitometric response of Hyperfilm beta-max (HFBM) to [14C]plastic standards was calibrated to tissue-equivalent concentrations of [125I]. Plastic sections with standard concentrations of [125I] and [14C] were apposed to HFBM for 1, 3, 5, and 9 days, with liver and muscle slices (20 microns thick) labeled with [125I]insulin. The relative optical densities (ROD = log 10 [1/gray level x 256(-1)] produced by [125I]- and [14C]plastic standards were converted to equivalent tissue [125I] concentrations (dpm/mm2). The response of HFBM to the [125I]- and [14C]plastic standards was similar (P less than 0.001). Standard curves of tissue [125I] (dpm/mm2) vs plastic [14C] (microCi/g) concentrations fit second order polynomials (r2 = 0.995-0.999). The results show that [14C]plastic standards are valid for measuring [125I] radioactivity in tissue slices by autoradiography with HFBM.     

Topographical assessment of accumulated radioactivity from [14C]2-deoxyglucose and [6(-14C)]glucose in rat forebrain at different survival periods

The uptake and retention of radioactivity was measured in discrete areas of rat brain at different times after i.v. injection of [14C]2-deoxyglucose or [6(-14)C]glucose, in unrestrained rats. In most brain regions, the accumulation of radioactivity from the two compounds was similar when a 30-min survival period for [6(-14)C]glucose was compared to a 45-min survival period for [14C]2-deoxyglucose. However, at those times, autoradiographic images of the hippocampus and piriform cortex appeared distinctly different for [14C]2-deoxyglucose and [6(-14)C]glucose. Relatively more radioactivity accumulated from [14C]2-deoxyglucose, compared to [14C]glucose, in the stratum lacunosum-moleculare of the hippocampus and in layer 4 of the isocortex. In contrast, relatively more radioactivity accumulated from [6(-14)C]glucose, compared to [14C]2-deoxyglucose, in the molecular and granule cell layers of the dentate gyrus, the CA1 pyramidal cell layer of the hippocampus, and in layer 2 of the piriform cortex. When rats were killed 5 min after injection of [6(-14)C]glucose, the relative neuroanatomical distribution of radioactivity was similar to the 30-min survival period, except in layer 4 of the isocortex, where relatively more radioactivity was present at the early time. When rats were killed 5 min after injection of [14C]2-deoxyglucose, in 20 of 24 brain regions examined, the absolute and relative amounts of accumulated radioactivity were similar when compared to that of the 45-min survival period. In contrast, the absolute and relative amounts of radioactivity were significantly greater for the 5-min compared to the 45-min survival period, in the CA1 pyramidal cell field, dentate gyrus, and layer 2 of the piriform cortex. For those regions, the appearance of autoradiograms prepared from rats killed 5 min after administration of [14C]2-deoxyglucose is remarkably similar to the appearance of autoradiograms prepared from rats killed 5 or 30 min after injection of [6(-14)C]glucose. Possible mechanisms are discussed to explain the observed differences in the accumulation of radioactivity in discrete brain regions after injection of [6(-14)C]glucose and [14C]2-deoxyglucose at the different survival times examined.