The induction of pulmonary phospholipidosis and the inhibition of lysosomal phospholipases by amiodarone

Administration of high doses of amiodarone to young adult rats leads to phospholipidosis of the lung, with extensive phospholipid storage by type II pneumonocytes and alveolar macrophages. Biochemical analysis reveals an increase in the total phospholipid content of the lung and in the proportion of phosphatidylcholine. The cause of the phospholipidosis is suggested to be the inhibition of lysosomal phospholipases, responsible for catabolizing phospholipids. It is shown that amiodarone is a potent inhibitor of phospholipases prepared from the soluble fraction of adult rabbit lung lysosomes.     

The induction of pulmonary phospholipidosis and the inhibition of lysosomal phospholipases by amiodarone.

Administration of high doses of amiodarone to young adult rats leads to phospholipidosis of the lung, with extensive phospholipid storage by type II pneumonocytes and alveolar macrophages. Biochemical analysis reveals an increase in the total phospholipid content of the lung and in the proportion of phosphatidylcholine. The cause of the phospholipidosis is suggested to be the inhibition of lysosomal phospholipases, responsible for catabolizing phospholipids. It is shown that amiodarone is a potent inhibitor of phospholipases prepared from the soluble fraction of adult rabbit lung lysosomes.     

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.     

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.     

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.     

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.     

The connections between the suprachiasmatic, ventrolateral geniculate and raphe nuclei studied by uptake of [14C]2-deoxyglucose

The [14C]2-deoxyglucose method was used to investigate the role of the ventrolateral geniculate and raphe nuclei in the control of the metabolism of the suprachiasmatic nuclei in adult female Wistar rats anaesthetized with alphaxalone. Three to seven days before the [14C]2-deoxyglucose studies a stimulating electrode was implanted or a lesion was made in the ventrolateral geniculate nucleus, or the ascending projection from the raphe nuclei was severed. Stimulation of the ventrolateral geniculate nucleus (biphasic rectangular pulses, 30 s on and 30 s off, 50 Hz, 500 microA pulse amplitude and 1 ms pulse duration) led to a significant increase in the relative metabolic activity of the ipsilateral suprachiasmatic nucleus and a smaller increase in the relative metabolic activity of the contralateral suprachiasmatic nucleus. The stimulus also increased significantly the relative metabolic activities of mainly the ipsilateral hypothalamus, midbrain central gray and reticular formation, all of which are too remote from the ventrolateral geniculate nucleus to be affected by current spread. In animals in which the ventrolateral geniculate nucleus had been lesioned, the relative metabolic activity of the suprachiasmatic nuclei was not significantly different from normal. In animals in which the ascending projection from the raphe nuclei had been severed, there was a slight, though significant increase in the relative metabolic activity of the suprachiasmatic nucleus of one side. These results, together with the effects of stimulating the suprachiasmatic nuclei [R. C. Maxwell and G. Fink, Neuroscience 23, 241-263 (1987)], show that the connections between the ventrolateral geniculate, raphe nuclei and suprachiasmatic nuclei are "metabolically functional", but that the integrity of the ventrolateral geniculate nucleus is not essential for maintaining the relative metabolic activity of the suprachiasmatic nuclei. The raphe nuclei may reduce the relative metabolic activity of the suprachiasmatic nucleus.     

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.     

Effect of isoproterenol on the uptake of [14C]glucose into glial cells

The effect of isoproterenol on the uptake of [14C]glucose into cultured glial cells was investigated in the present study. Isoproterenol markedly stimulated the uptake of [14C]glucose 30 min after incubation. This action was produced in a dose-dependent manner. A positive correlation between the increase of [14C]glucose uptake and the stimulation of adenylate cyclase induced by isoproterenol was obtained (r = 0.99). The effect of isoproterenol was reduced by the beta-adrenergic blockers but not by other blockers, indicating the selective action of isoproterenol. The results obtained suggest that isoproterenol stimulates the uptake of glucose into glial cells through the activation of beta-adrenoceptors which are linked to adenylate cyclase.     

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.     

A possible neuronal release of [14C] GABA from the rat cerebellum in vivo

Release patterns for exogenously applied [¹⁴C] labelled -amino-n-butyric-acid (GABA) have been investigated in rat cerebellar cortex in vivo. An increase in [¹⁴C] GABA release could be evoked by stimulating with high (40 mM) K⁺ or veratridine (10^–4 M) but not with direct electrical stimulation. Biphasic patterns for high K⁺ and possibly veratridine stimulated release of GABA suggest the existence of two separate anatomical sources of isotope which are sensitive to these depolarising stimuli. Both K⁺ and veratridine-evoked GABA release are calcium dependent. Studies involving partial replacement of Na⁺ with HEPES, (N-2-hydroxyethyl-piperazine-N-2-ethane-sulphonic acid), sucrose or choline chloride also reveal a sodium dependency of [¹⁴C] GABA release. These studies collectively indicate a neuronal source for evoked GABA release, a criterion for transmitter identification not previously satisfied in the cerebellar cortex.Supported in part by a grant from S.R.C. to N.D. and C.G.D.Supported by CAPES and FAPESP (Brazil)Supported by CNPq and FAPESP (Brazil)     

The effect of chronic ethanol consumption on [C]deoxyglucose uptake in rat brain in vivo

The uptake of [¹⁴C]deoxyglucose by brains of rats that were given alcohol in drinking water for 7 months was investigated. There was a general, approximately 50%, increase in deoxyglucose uptake in brains of ethanol-treated rats with areas of the limbic system being particularly affected.     

Sialylation of Thomsen-Friedenreich (T) and blood type NM antigens by transferases in human sera measured by [14C]NAN uptake

Human erythrocyte T & NN antigens were exposed to fresh sera from single human donors and CMP-[14C]sialic acid without activators in a single 24 hr incubation at 37 degrees C. There was similar sialic acid (NAN) uptake, on the average 2.7 mol/mol T antigen subunit (mol wt 40,000), with various NN- and MM-derived T antigens and transferase sera in all different combinations. Sera from donors with the M gene but not from those lacking it incorporated NAN into NN antigen (c. 1 mol NAN/mol NN antigen subunit, mol wt 50,000). The profound difference in sialyltransferase action between sera from donors with the M gene and those lacking it was substantiated by repeated incubations of NN-derived T antigen with CMP-NAN and MM donor transferase serum, which incorporated 32% more NAN than did repeated incubations of the same antigen with CMP-NAN and NN donor transferase serum. The greater NAN incorporation by sera of donors with the M gene is due to an additional transferase-modifier substance present only in persons possessing the M gene.     

In vivo incorporation of exogenous [1-14C]stearic acid into neurons and astrocytes

Exogenous stearic acid is needed to synthesize the membranes of neurons and astrocytes. Subcutaneously injected [1-14C]acid is taken up through the 'blood brain barrier' and incorporated into lipids of both cell types, the specific radioactivity being higher in astrocytes as compared to neurons (2200 and 800 cpm/mg proteins, respectively), 20 h after injection. Phospholipids contain high amount of radioactivity (80% in astrocytes, 65% in neurons); glycosphingolipids contain low quantities of label in the two cell types. The injected acid is partly metabolized in the brain by elongation and desaturation (thus providing very long chains, saturated mono-unsaturated and poly-unsaturated); it is also partly degraded into acetate units (utilized for synthesis of palmitic acid).     

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)     

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.