Factors influencing the uptake and loss of radiosulfate by rat renal cortical tissue in vitro

Rat kidney cortical slices, during incubation in vitro, lose previously accumulated radiosulfur when exposed to conditions (e.g. addition to the medium of metabolic inhibitors) which normally depress the uptake of S³⁵. The extent of this loss is not affected significantly by the presence of phlorhizin, an agent which enhances markedly radiosulfate accumulation. On the other hand, when tissues are chilled to 1°C., loss is slight or negligible even in the presence of metabolic inhibitors. These data, and observations on the effect of pre-incubation of kidney slices in S³⁵-free media before the addition of radiosulfate, have been interpreted as evidence that S³⁵ accumulation in vitro may be resolved into at least two processes, namely (a) entrance of the isotope-labelled anion into the cells, by diffusion and/or active transport, and (b) complexing of S³⁵ (in ionic or other form) with an intracellular component. The postulated complex is stabilized, perhaps through inactivation of a specific enzyme, by chilling the tissue to 1°C. Possible relationships are discussed among the observations noted above, sulfur metabolism in general, and aspects of the known in vivo transport mechanism for sulfate ion; i.e., renal tubular reabsorption.     

The Effect of Temperature on the Uptake of Radiosulfate by Rat Renal Tissue from Radiosulfate-Containing Solutions in Vitro

Kidney cortex slices incubated in vitro at 0°C. accumulate radiosulfate from the incubation medium. This process differs from the previously described uptake of radiosulfate by renal tissue incubated at 38°C., for instance, in the lesser sensitivity of the uptake at 0°C. to differential effects of Na⁺ as compared with K⁺ ions, and of sucrose as compared with glucose. Phlorizin inhibits radiosulfate accumulation at 0°C., whereas it enhances the uptake at 38°C. Effects of the cations K⁺ and Na⁺ and of phlorizin at temperatures intermediate between 0° and 38°C. have been studied. Parallels have been noted between the accumulative processes for radiosulfate of kidney slices maintained at 0°C. and of mitochondria isolated from rat liver and kidney cortex. These similarities may be attributed to an important role of radiosulfate uptake by mitochondria in slice accumulation of radiosulfate in the cold.     

The accumulation and metabolism of [C]hypoxanthine by slices of rabbit renal medulla

The uptake of hypoxanthine by rabbit renal medulla has been studied with in vitro conditions. Unlike the uptake by renal cortex slices reported earlier, no evidence was found for involvement of an organic cation transport system. Medullary accumulation of the ¹⁴C-labeled material occurred in the absence of O₂ if glucose was present as substrate. Uptake of the ¹⁴C label was not supported by other sugars or metabolic intermediates, however. Metabolic inhibitors reduced both aerobic and anaerobic uptake. Tissue extracts were subjected to high-voltage electrophoresis and gel filtration for identification of possible metabolites. These studies indicated that most of the uptake of ¹⁴C-labeled material was accounted for by its conversion to inosine-like and/or inosinic acid-like compounds. That is, when experimental conditions were designed to retard slice metabolism of hypoxanthine, tissue to medium ratios for ¹⁴C approximated 1.0. A third metabolite was found occasionally, but remains unidentified.     

Energy-linked Sulfate Uptake by Corn Mitochondria via the Phosphate Transporter

Corn shoot mitochondria possess an energy-linked transport system for sulfate uptake as demonstrated by osmotic swelling and [³⁵S]SO₄²⁻ accumulation. Maximum uptake is secured in the presence of Mg²⁺ and oligomycin with sucrose for osmotic support. Neither phosphate nor dicarboxylate anions are required. When added simultaneously, millimolar concentrations of phosphate block [³⁵S]SO₄²⁻ uptake after the initial minute. Mersalyl, N-ethylmaleimide, and 2,4-dinitrophenol are strong inhibitors of sulfate uptake; n-butylmalonate is a weak inhibitor. These inhibitors act in the same fashion on phosphate uptake. It is concluded that sulfate uptake in the absence of phosphate is by the phosphate transporter.     

THE EFFECTS OF FLUORIDE FEEDING ON THE ORGANIC MATRIX OF BONES AND TEETH OF PIGS AS OBSERVED BY AUTORADIOGRAPHY AFTER IN VITRO UPTAKE OF CA45 AND S35

Demineralized sections of fluorinated bones and teeth have been studied by autoradiography following in vitro uptake of Ca⁴⁵ or S³⁵O₄. The portions of tissue which do not become mineralized (cartilage, prebone, predentine, and precementum) show an increased Ca⁴⁵ uptake apparently related to an increase in chondroitin sulfate content in fluorosis. The tissues from the fluoride-fed animals show an increase of in vitro uptake of sulfur in the tissues which become mineralized (bone, dentine, cementum).     

ON THE SITE OF SULFATION IN COLONIC GOBLET CELLS

The location of bound S³⁵ in the goblet cell of the rat colon at time points from 2 to 60 minutes after administration of S³⁵ as sodium sulfate has been observed in vivo and in vitro by radioautographic techniques. Grains were first observed by electron microscopy over the stacked lamellae of the paranuclear part of the Golgi apparatus. The label was subsequently found associated with the supranuclear Golgi lamellae and was then seen associated with the smooth membranes limiting the mucin granules in the goblet. Finally, between ½ and 1 hour, the secreted mucus product in the crypts became radioactive. Neither mitochondria nor the endoplasmic reticulum was labeled. It is concluded that the Golgi apparatus is the organelle in which sulfation occurs.     

The reduction of inorganic sulphate to inorganic sulphite in the small intestine of the rat

1. Whole scrapings of rat intestinal mucosa were incubated with carrier-free sodium [³⁵S]sulphate. Radioactivity was found in S-sulphocysteine and to a small extent in S-sulphoglutathione. 2. Whole scrapings of rat intestinal mucosa incubated with carrier-free sodium [³⁵S]sulphate and oxidized glutathione formed S[³⁵S]-sulphoglutathione as the main radioactive product. The amount of S[³⁵S]-sulphocysteine formed was considerably lower than in a control that contained no oxidized glutathione. 3. The supernatant fraction of homogenates of rat intestinal mucosa catalyses the NADPH-dependent reduction of adenosine 3′-phosphate 5′-sulphatophosphate to inorganic sulphite. NADH or GSH fail to replace NADPH as reducing agents. 4. The formation of inorganic [³⁵S]sulphite from inorganic [³⁵S]-sulphate may account for the incorporation of [³⁵S]sulphate into S-sulphoglutathione by the small intestine of the rat in vivo and in vitro.     

Role of o-acetylserine in hydrogen sulfide emission from pumpkin leaves in response to sulfate

In the presence of excess sulfate, cysteine synthesis in pumpkin (Cucurbita pepo) leaves is not limited by sulfate reduction, but by the availability of O-acetylserine. Feeding of O-acetylserine or its metabolic precursors S-acetyl-coenzyme-A and coenzyme A to leaf discs enhanced the incorportion of [³⁵S]sulfate into reduced sulfur compounds, mainly into cysteine, at the cost of lowered H₂S emission; the uptake and reduction of sulfate is not affected by these treatments. β-Fluoropyruvate, an inhibitor of the generation of S-acetyl-coenzyme A via pyruvate dehydrogenase, stimulated H₂S emission in response to sulfate. This stimulation is overcompensated by addition of O-acetylserine, S-acetyl-coenzyme A, or coenzyme A. These results indicate that, in the presence of high amounts of sulfate, excess sulfur is reduced and emitted as H₂S into the atmosphere. The H₂S emitted seems to be produced by liberation from a precursor of cysteine rather than by cysteine desulfhydration.     

Sulfate transport by mouse renal cortical slices does not represent uptake by brush-border membrane

We measured uptake of isotopically ³⁵S-labelled sulfate anion by slices and by brush-border membrane vesicles prepared from mouse renal cortex to identify: (i) whether metabolic incorporation of anion influences net transport; (ii) which membrane is primarily exposed in the renal cortex slice. Slices accumulated sulfate without significant incorporatoin into metabolic pools. Net uptake of sulfate at 0.1 mM by the slice occurred against an electrochemical gradient as determined by mesurement of free intracellular sulfate concentration, the isotopic distribution ratio at steady-state, and the distribution of lipophilic ions (TPP⁺ and SCN^?). Carrier mediation of sulfate transport in the slice was confirmed by observing concentration-dependent saturation of net uptake and counter-transport stimulation of efflux. Anion uptake was Na⁺-independent, K⁺- and H⁺-stimulated, and inhibited by disulfonated stilbenes. Brush-border membrane vesicles accumulated sulfate by a saturable mechanism dependent on a Na⁺ gradient (outside > inside); others have shown that uptake of sulfate by brush-border membrane vesicles is insensitive to inhibition by disulfonated stilbenes. These findings indicate that different mechanisms serve sulfate transport in renal cortex slice and brush-border membrane vesicle preparations. They also imply that the slice exposes an epithelial surface different from the brush-border, presumably the basolateral membrane, or its equivalent, since sulfate transport by slices resembles that obserbed with isolated basolateral membrane vesicles.     

Incorporation and Rate of Loss of S35-Methionine by Adult Rat Trachea in Organ Cultures and in Living Animals

Full-thickness pieces of adult rat trachea were supported on rayon on the surface of clotted medium in watch glasses. Differentiated epithelium was reduced in height during 25 days of cultivation because basal cells and some columnar cells migrated to cover exposed parts of the explants and because some differentiated cells died and were shed. S³⁵-methionine was (a) placed on explants in vitro and (b) injected intraperitoneally in living rats. Cultured tissues and tissues of living rats were examined by autoradiography at 4 and 24 hours and 4, 7, and 11 days after labeling. Although migratory undifferentiated epithelial cells appeared in cultured trachea, all living epithelial cells in vitro incorporated and subsequently lost S³⁵-methionine to the same extent as did epithelium of intact rats. The biologic half-life of methionine in rat tracheal epithelium in vivo and in vitro was about 5 days.     

Incorporation of [S]Sulfate and [C]Bicarbonate into Karyotype-specific Polysaccharides of Chondrus crispus

Gametophytic and sporophytic tissues of Chondrus crispus (Stackhouse) cultured in vitro were labeled with ³⁵S and ¹⁴C. The major sulfated polysccharides isolated from the two karyotypes were characterized by KCI fractionation, immunoprecipitation, and infrared spectroscopy. Reproducibility of data has been demonstrated by an experiment using gametophytic T₄ strain with five replicates per time point. The rate of sulfate uptake was similar in haploid and diploid plants from a given area cultured for a similar time. Cultures from different sources cultured for different times showed different uptake and incorporation levels. Although sulfate uptake did not appear to be karyotype-related, the pattern of incorporation of ³⁵S and ¹⁴C into polysaccharides was ploidy-specific.     

Relationship between binding activity of 67Ga and low sulfated acid glycosaminoglycans

The sulfate content of acid glycosaminoglycan (AGAG) extracted from granuloma which had been produced by turpentine oil was inversely proportional to the amount of ⁶⁷Ga accumulation in the granuloma. Additionally, the lowest sulfation occurred in granuloma at a peak of inflammation when the uptake of ⁶⁷Ga had reached a maximum. On the basis of electrophoretic pattern, sulfate content, and specific optical rotation, it was concluded that acid glycosaminoglycans obtained from granuloma are mainly composed of chondroitin sulfate-A, -B, and desulfated heparin, while heparan sulfate was a minor component. From in vitro assays, desulfated acid glycosaminoglycans, especially desulfated-heparin and desulfated-heparan sulfate, were found to have a high affinity to ⁶⁷Ga. These results suggest that low- or de-sulfation of AGAG is related to the accumulation of ⁶⁷Ga in inflammatory lesions such as granuloma. Moreover, these results suggest that ⁶⁷Ga does not bind to glycosaminoglycans via sulfuric acid residues.     

AUTORADIOGRAPHIC STUDIES OF THE UTILIZATION OF S35-SULFATE BY THE CHICK EMBRYO

From studies of autoradiograms of various developmental stages of the chick embryo containing S³⁵ given us sulfate it was determined that as early as Stages 3+ and 4 there is a selective utilization or accumulation of sulfate by the various parts. The earliest accumulation site is the axial portion of the primitive streak and the floor of the groove. Later S³⁵ was found in the head process, Hensen's node, notochord, amniocardiac vesicle, wall of the omphalomesenteric vein, endocardium, subendocardial jelly, mesenchyme destined to become cartilage, basement membrane area of the gut, and a mucopolysaccharide layer formed on the free surface of the stomach. The early notochordal localizations of S³⁵ coincide with the region in which a thin ring of chondroitin sulfate is subsequently laid down. However, it is apparent that there is an intracellular accumulation of inorganic sulfate by the chondroitin-forming cells prior to the time they produce sufficient chondroitin sulfate to be demonstrable histochemically. It was interesting to note that the endocardium appears to concentrate sulfate that later apparently finds its way into the subendocardial jelly. The fact that those mesenchymal cells which later form chondroblasts begin to utilize sulfate selectively before histological differentiation is apparent was determined. In addition, the presence of sulfate-containing substances in the forming basement membrane of the gut would seem to indicate that sulfate is important in the histological differentiation of this membrane.     

Acid mucopolysaccharide synthesis in the secondary palate of the developing rat at the time of rotation and fusion

The amount of hexosamines and acid mucopolysaccharides present in the rat secondary palate increases during the critical stages of palatogenesis, namely, rotation and fusion. The synthesis of acid mucopolysaccharides in vivo and in vitro in the palate was determined by the incorporation of ³H-glucosamine and Na₂S³⁵O₄. The labeled mucopolysaccharides were isolated by DEAE-cellulose chromatography and were identified on the basis of several criteria as hyaluronic acid and sulfated acid mucopolysaccharides. Hyaluronic acid accounted for approximately 60% of the total acid mucopolysaccharides synthesized in the palate both in vivo and in vitro. DON (6-diazo-5-oxonorleucine), a known inhibitor of acid mucopolysaccharide synthesis, inhibited the incorporation of ³H-glucosamine and Na₂S³⁵O₄ by palatal shelves in vitro by 70%.     

Uptake of taurine into subcellular fractions of C-6 glioma cells.

Subcellular fractions from cultured C-6 glioma cells prepared by methods similar to those for crude synaptosomal fractions of rat cerebral cortex accumulated [³⁵S]taurine as did intact glioma cells. Thus, the accumulation of taurine was dependent on temperature and sodium concentration and sensitive to osmotic shock. The kinetic properties of this uptake are characterized by an apparent K_m, of about 25 μm, The properties of taurine uptake into subcellular fractions from C-6 glioma cells were compared with those of crude synaptosomal fractions and differences could be observed in temperature sensitivity and with metabolic inhibitors, which were less potent in the glioma preparation. Equilibrium density gradient centrifugation of subcellular fractions from glioma cells revealed that particles containing [³⁵S]taurine sediment to a lower buoyant density than mitochondria. But on co-sedimentation of subcellular fractions from glioma cells with synaptosomal fractions derived from cerebral cortex, differences in the buoyant density between these two preparations could be found. The findings support the possibility of a contamination of synaptosomal fractions with subcellular fractions derived from glial origin.     

Differences between substrate specificities of l-glutamate uptake by neurons and glia,studied in cell lines and primary cultures

High affinity uptake of [³H]l-glutamate was studied in cultures of continuous cell lines, originating either from mouse neuroblastoma or rat glioma, and in two types of primary cultures containing cerebellar granule cells and astrocytes from cerebral cortex, respectively. In the continuous lines, d- and l-aspartate-4-hydroxamate were found to interact preferentially with the uptake of [³H]l-glutamate in glioma cells while l-glutamate-5-hydroxamate and 2-aminoadipate interacted more strongly with [³H]l-glutamate uptake in neuroblastoma cells, d-Aspartate-4-hydroxyamate, l-glutamate-5-hydroxamate and 2-aminoadipate were inactive as inhibitors of [³H]l-glutamate uptake by either granule cells or astrocytes, grown in primary culture, but several other glutamate analogues, which did not differentiate between neuroblastomal and gliomal uptake of [³H]l-glutamate, were somewhat stronger inhibitors of [³H]l-glutamate uptake in astrocytes as compared to that in granule cells. However, all of these compounds (N-acetyl-l-glutamate, formimino-l-aspartate, d-homocysteate, l-homocysteate and dl-2-methylglutamate) were only very weak inhibitors and, consequently, it is unlikely that any of them could be useful in experiments with central nervous tissue in vivo or, at least, in brain slices in vitro, attempting to resolve the uptake of l-glutamate into glia- and neuron-localized components.     

AUTORADIOGRAPHIC DETERMINATION OF S35 IN TISSUES AFTER INJECTION OF METHIONINE-S35 AND SODIUM SULFATE-S35

The loss of "bound" S³⁵ that occurs during various mounting procedures used in autoradiography was studied in healing surface wounds of rats treated with either methionine-S³⁵ or Na₂S³⁵O₄. Valid autoradiography of bound S³⁵ in this tissue is not possible until 48 hours after radiosulfate and 24 hours after radiomethionine injection, when the S³⁵ is almost entirely bound in large protein and polysaccharide molecules. Autoradiograms of S³⁵ given in both the organic and inorganic form reveal substantial over-all loss of the bound isotope from sections subjected to contact with solvents prior to autoradiography. A comparison of autoradiograms prepared by dry-mounting sections of frozen-dried tissue with autoradiograms of wet-mounted sections of the same tissue suggest that the loss is proportional to the extent of the contact with solvents. Evidence suggests that loss of the isotope occurs during contact of the ribbon or section itself with solutions after fixation and cutting and prior to radiation exposure. No appreciable loss of the bound isotope seems to occur during contact of the intact tissue specimen with a variety of fluid fixatives except for a marginal zone at the excision edges of the tissue. The potential hazard of displacement of the isotope during fixation, however, remains. Technics which prevent loss of the isotope and fogging of the nuclear emulsion permit the use of thinner sections and emulsion films and the fine resolution of image rendered possible by the physical properties of S³⁵.     

Histochemical and Autoradiographic Studies on the Effects of Aging on the Mucopolysaccharides of the Periosteum

An autoradiographic study was made using S³⁵-sulfate for the localization, distribution, and variation in the mucopolysaccharide content of the femoral periosteum of rats from birth to old age. The mucopolysaccharides were also studied histochemically, using toluidine blue O, Rinehart and Abu'l-Haj's colloidal iron method, and the periodic acid-Schiff reaction, before and after hyaluronidase treatment. Autoradiograms revealed the uptake of S³⁵ particularly in the vicinity of the preosseous zone and adjacent osteoblasts. This labelling was highest at the period of rapid bone growth. With increasing age, the S³⁵ uptake became progressively less. The preosseous zone showed γ-metachromatic staining at all ages after treatment with toluidine blue. Active osteoblasts were mostly orthochromatic, however, β-metachromasia was exhibited at a later age. Abundant amounts of intra- and extracellular mucopolysaccharides of both the acid and neutral type were demonstrated in the periosteum. S³⁵ uptake and γ-metachromasia show the presence of sulfated mucopolysaccharides, of which chondroitin sulfate predominates in the preosseous zone. Since S³⁵ uptake is high in active osteoblasts, the inability to demonstrate metachromasia in osteoblasts may indicate either that chondroitin sulfate is liberated as fast as it is being produced, or that it may be present within the cells in a precursor form not detectable by histochemical methods.     

Active and Passive Components of Sulfate Uptake in Sunflower Plants

The aim of the investigation was to identify components of active and passive ion uptake and transport in roots of plants and to assess their quantitative relations under different external and internal conditions. The uptake of radiosulfate and water by young sunflower plants from complete nutrient solutions labelled with ³⁵S was studied. The metabolism-linked nature of the sulfate uptake in the root following the passive migration into the apparent free space (AFS) was demonstrated by the addition of sodium. selenate, 2,4-dinitrophenol, potassium cyanide, and sodium azide to the nutrient solutions. The magnitude of the AFS measured on a root volume basis varied between 14 and 57 per cent depending on the pretreatment of the plants and the sulfate concentration of the nutrient solution. The variations were supposed to be due to different capacity to bind sulfate by exchange-adsorption within the AFS. The amounts of sulfate in different fractions of the total AFS-uptake were computed under certain theoretical assumptions. A quantitative connection was proposed between the magnitude of the adsorbed sulfate fraction in the AFS and the rate of active uptake into the symplasm. The exchange-adsorption probably constitutes the initial stage of active ion uptake. The stimulating effect by water on ion uptake would be an increase of the speed of transporting ions to, from, or along the adsorption sites in the AFS. Experiments conducted at temperatures in the nutrient solution between 5 and 35 C elucidated the multistep nature of ion transport within a root.     

Synechococcus elongatus PCC 7942 is more tolerant to chromate as compared to Synechocystis sp. PCC 6803

Two unicellular cyanobacteria Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942 showed contrasting responses to chromate stress with EC₅₀ of 12 ± 2 and 150 ± 15 μM potassium dichromate respectively. There was no depletion of chromate in growth medium in both the cases. Using labeled chromate, very low accumulation (<1 nmol/10⁸ cells) was observed in Synechocystis after incubation for 24 h in light. No accumulation of chromate could be observed in Synechococcus under these conditions. Chromate oxyanion is known to enter the cells using sulfate uptake channels. Therefore, inhibition of sulfate uptake caused by chromate was monitored using ³⁵S labeled sulfate. IC₅₀ values of chromate for ³⁵sulfate uptake were higher in Synechococcus as compared to Synechocystis. The results suggested that the sulfate transporters in Synechococcus have lower affinity to chromate than those from Synechocystis possibly due to differences in affinity of sulfate receptors for chromate. Bioinformatic analyses revealed presence of sulfate and chromate transporters with considerable similarity; however, minor differences in these may play a role in their differential response to chromate. In both cases the IC₅₀ values decreased when sulfate concentration was reduced in the medium indicating competitive inhibition of sulfate uptake by chromate. Interestingly, Synechococcus showed stimulation of growth at concentrations of chromate less than 100 μM, which affected its cell size without disturbing the ultrastructure and thylakoid organization. In Synechocystis, growth with 12 μM potassium dichromate damaged the ultrastructure and thylakoid organization with slight elongation of the cells. The results suggested that Synechococcus possesses efficient strategies to prevent entry and to remove chromate from the cell as compared to Synechocystis. This is the first time a differential response of Synechococcus 7942 and Synechocystis 6803 to chromate is reported. The contrasting characteristics observed in the two cyanobacteria will be useful in understanding the basis of resistance or susceptibility to chromate.