Increase In 32P Uptake Research Articles

1,25-dihydroxyvitamin D3 stimulates vesicular transport within 5 s in polarized intestinal epithelial cells

Controversy remains regarding whether the seco-steroid hormone 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) enhances calcium and phosphate movement across the intestinal epithelial cell by facilitated diffusion or a vesicular transport mechanism. In this study we investigated whether membrane trafficking, as judged by confocal microscopy, was sufficiently rapid in comparison to hormone-stimulated uptake of phosphate (32P). Primary cultures of chick intestinal cells were established overnight either in Petri dishes (uptake studies) or chambered coverslips (confocal microscopy). Addition of 130 pM 1,25(OH)2D3 resulted in an apparent increase in 32P uptake within 1 min, relative to controls, that was statistically significant from 3-10 min of incubation. Using the endocytic marker dye, FM1-43, confocal microscopy revealed a profound decrease in membrane-associated fluorescence (apical> basal) within 10 s of hormone treatment, a return of fluorescence at 15-65 s, followed by another round of decreasing and increasing fluorescence. Between 3-9 min of incubation, fluorescence intensity increased 50% (apical region) and 20% (basal region) over control conditions. An antibody (Ab 099) directed against a putative membrane receptor for 1,25(OH)2D3 (1,25D3-MARRS) inhibited both 32P uptake, and changes in fluorescence. In addition, the protein kinase C (PKC) inhibitor, calphostin C, inhibited both 32P uptake and the observed 1,25(OH)2D3-mediated changes in fluorescence. At the microscopic level, calphostin C pretreatment abolished the very rapid redistribution of the endocytic marker dye, although a slight increase in fluorescence was still observed. We conclude that 1,25(OH)2D3-stimulated vesicular trafficking is mediated by the 1,25D3-MARRS protein, implicates a PKC signaling mechanism, and occurs in a time frame that is commensurate with a role in ion transport.

Growth on Indium-Tin Oxide-Coated Glass Enhances 32P-Phosphate Uptake and Protein Labelling of Adherent Cells

ABSTRACT A method to improve the efficiency of labelling of adherent cells with radioactive 32P is described. Cells are grown on a glass surface which is coated with indium-tin oxide, a commercially available, transparent material which permits excellent cell adhesion and growth. The results show that a 2 to 3-fold increase in 32P uptake by the cells can be achieved by growing cells on this material, compared to conventional tissue culture plastic.

Impairments in hepatocyte phosphoinositide metabolism in endotoxemia

The status of phospholipid metabolism and inositol lipids-mediated transmembrane signaling in rat hepatocytes was analyzed during chronic, nonlethal endotoxemia. Rats were infused intravenously (IV) with Escherichia coli endotoxin (ET) via subcutaneously implanted osmotic pumps at a rate of 0.1 mg 100 g bw/day . The experiments were performed after 30 hours of ET or sterile saline (NaCl) infusion, in hepatocytes prelabelled “in vitro” with 32P (15 μCi/mL) and further stimulated with vasopressin (VP, 0.23 μmol/L). Similar experiments were done with food-restricted animals, whose food intake was matched with the voluntary intake of ET-infused rats. Uptake of 32P label into phosphatidic acid (PA), phosphatidylinositol 4-phosphate (PIP), and phosphatidylinositol 4,5-bisphosphate (PIP 2) occurs rapidly in cells from pair-fed, saline and ET-infused animals, and reaches a plateau between 60 and 80 minutes of incubation. Labeling of phosphatidylinositol (PI), phosphatidylethanolamine (PE), and phosphatidylcholine (PC) proceeds linearly after a ten-minute lag period for PI and 20 minutes for the two other lipids. The nutritional state greatly affects the distribution of 32P uptake into lipids, resulting in very low labeling of PA and PI and a high labeling of poly-PI as compared with control (taken from untreated rats) cells. In ET- v saline-infused rats, the labeling of PI and PE was depressed concomitantly with a proportional increase in the labeling of PIP and PC. The ability of VP to induce polyphosphoinositide (poly-PI) degradation in hepatocytes from saline-infused animals was similar to that observed in control cells. The rapid loss in PIP 2 and PIP labeling (50% and 35%, respectively, within 30 seconds) was paralleled by an increase in 32P uptake in PA and followed by a later increase in PI labeling (150% at five minutes), and a sustained labeling of poly-PI towards prestimulation levels. In ET-infused rats, the early poly-PI response to VP stimulation was inhibited by 40% with no subsequent recovery in the PIP 2 labeling up to the last sampling time (five minutes). Also, the stimulation of PI turnover was 50% lower than that observed in saline-infused animals. The well-known stimulating effect of VP on 32P uptake into PI and PA when present from the start of incubation of the cells with the precursor, was abolished in ET-infused rats, while the labeling of PIP 2 was inhibited by 50%. In cells of saline-infused animals, 32P uptake into PA and PI was greatly increased, as in hepatocytes from control and pair-fed animals, with no effect at the level of poly-PI labeling. The results support the concept that ET infusion significantly perturbs changes in inositol lipid metabolism elicited by VP, affecting PIP 2 degradation and resynthesis at the plasma membrane level and PI turnover and/or synthesis in the endoplasmic reticulum.

Erythrocyte membrane phosphorylation in aging rats.

By sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the protein profile and autophosphorylation of plasma membranes of red cells from 3-, 9-, 18-, and 30-month-old rats was examined. No major changes in SDS-PAGE protein profiles were noticeable. However, endogenous (auto) and exogenous phosphorylation in the presence of (gamma-32P)ATP showed an age-dependent increase in 32P uptake. pH optimum was between 8.0 and 9.0 for all age groups. Cyclic AMP was without effect on either endogenous or exogenous phosphorylation. Further analysis of the autophosphorylation reaction by quantification of autoradiograms obtained from SDS-PAGE gels displayed an age-dependent increase in phosphorylation of bands 2, 3, 4.1, and 4.5 [nomenclature of Fairbanks et al. Biochemistry 10:2606, 1971] and of several other minor bands. The data imply that alteration in red cell membranes may occur during aging of rats causing an enhancement in endogenous kinase activity and possibly in the number of phosphate acceptor sites as well.

Radioactive phosphorus-uptake-testing variables before and after enucleation.

Values for radioactive phosphorus uptake from 23 eyes with malignant melanomas of the choroid were compared before and after enucleation. In all but two eyes, the percentage increase in 32P uptake was higher immediately after enucleation than before enucleation. In 11 eyes, the variation was more than 100%. In one eye, the percentage increase of 32P uptake was 68% before enucleation and 137% after enucleation. The clinical importance of our findings is that pressure on the globe by the detector probe before enucleation may locally decrease choroidal blood volume thereby causing an increase in the calculated 32P uptake.

A study of the kinetics of the muscarinic effect on phosphatidylinositol and phosphatidic acid metabolism in rat brain synaptosomes.

The uptake of [32P]phosphate into phosphatidylinositol and phosphatidate was measured in synaptosomes incubated in Krebs-Ringer bicarbonate buffer, pH7.4. The apparent dissociation constants for acetylcholine and carbamoylcholine was estimated from the increase in 32P uptake caused by these agents. These apparent constants were similar for both phosphatidylinositol and phosphatidate and were 2.7 +/- 0.5 MICROmeter for acetylcholine and 12 +/- 2 micrometer for carbamoylcholine when Ca2+ concentration was 0.75 mM. Under the same conditions the inhibition of the carbamoylcholine-induced increase in 32P uptake, caused by atropine, is consistent with atropine being a competitive inhibitor, with an apparent inhibition constant of 0.35 +/- 0.05 micrometer. The apparent constants were dependent on the Ca2+ concentration, and were greater in 2.54 mM-Ca2+. The former values for the kinetic constants are similar to the muscarinic-receptor dissociation constant, which indicates that the binding of the agonist to the receptor may be rate-limiting in this series of reactions when the Ca2+ concentration is 0.75 mM.

Early increase in the phosphorylation of liver chromatin non-histone proteins from thyroidectomized rats treated with triiodothyronine.

The effect of triiodothyronine (T3) on the endogenous phosphorylation of chromatin nonhistone proteins (NHP) was investigated. Chromatin NHP were extracted from livers of thyroidectomized rats treated with T3 (30 μg/100 g BW) for varying times, and the preparations were then incubated in vitro in a medium containing [γ-32P]ATP. Moreover, because of the complex heterogeneity of these proteins, we analyzed by disc gel electrophoresis the 32P uptake of the individual major classes of phosphorylated NHP. A marked increase in 32P uptake was found not only in chromatin NHP prepared from rats treated with T3 for 2 or 4 consecutive days, but also in NHP preparations obtained from rats sacrificed 3 h after a single injection of T3, accounting for an early effect of the thyroid hormone. Electrophoretic fractionation of the phosphorylated NHP obtained from T3 treated rats provided evidence that for the most effective hormonal treatments, the increased radioisotope uptake into NHP was more evident in some protein fra...

On the Mode of Activation of Sequestered Messengers in Artemia salina

Activation of the dormant embryos of Artemia salina was marked by a rapid increase in 32P uptake which reached a stationary phase after 6 h of activation. The increase in 32P uptake by whole cysts was paralleled by its incorporation into nucleotides. Fractionation of acid-soluble nucleotides and alkaline hydrolysate of nucleic acids on Dowex-1-formate column revealed the 32P radioactivity to be exclusively localised in AMP. Analysis of the labelled RNA species extracted at different stages of development indicated a preferential labelling of small molecular weight species till the emergence of the embryos, followed by the de novo synthesis of messenger and stable RNA species in later stages of development. During early development, polyadenylated RNA species were localised in the particulate fraction sedimenting at 16,000 rpm and their location shifted to the soluble fraction as development proceeded. Activation of performed messengers by phosphorylation of the adenylate residue of their poly A stretches and translocation of the capacitated messengers to the cytosol via a RNP-membrane complex is proposed as a trigger of embryonic differentiation.

Photosynthetic activity of vegetable plants and its horticultural significance

This study was undertaken to elucidate the photosynthetic activity, carbohydrate metabolism, translocation of assimilates and mineral nutrient uptake in young tomato plants at very low carbon dioxide concentrations.Radioisotopes of 14CO2, H332PO4 and 86RbCl were used as tracers under controlled conditions. Photosynthetic rates were also measured with gas exchange method by means of assimilation chambers and an infrared gas analyser.Photosynthetic rate of tomato leaf was linearly proportional to the CO2 concentration within the range from 70 to 300ppm CO2 at three light intensities of 0.07, 0.16 and 0.53 cal•cm-2•min-1. In an atmosphere containing 80ppm CO2, total activity of 14C fixed into the plants dropped to 35 per cent of the normal, and at the same time significant decrease was observed in the translocation of 14C-assimilates from the leaves to the roots.Fairly good amount of 14C-compounds, fixed in the evening and remained in the leaf till next morning, was translocated into the roots under normal photosynthetic conditions. In a CO2-deficient atmosphere, however, a downward translocation of 14C-compounds was slightly restricted and respiratory losses of 14C-compounds were considerably increased in the leaf.Tomato plants placed in an atmosphere containing 60ppm CO2 for 2 hours had lower level of carbohydrates in the leaf and root.A remarkable increase in 32P uptake was found at very low CO2 concentration in the tomato plants which had been illuminated for 6 hours before 32P-treatment. In contrast, the CO2 depletion caused marked decrease in the 32P uptake for the unlighted plants.The uptake of 86Rb was considerably restricted at low CO2 concentration both in the lighted and unlighted plants. The absorption and accumulation of rubidium were highly dependent on the photosynthetic activity in the tomato plants.From these results, the relationship between the translocation of assimilates and the uptake of mineral nutrients was discussed from physiological point of view.

Lutenizing hormone bioassay based on uptake of radioactive phosphorus by the chick tests

1. 1. Neuraminidase, a bacterial enzyme, was used to selectively destroy follicle-stimulating hormone (FSH) activity of chicken anterior pituitary glands. 2. 2. The enzyme-treated pituitary material and various other pituitary hormones were tested to determine their effect on 32P uptake by chick testes. Adrenal cortical stimulating hormone (ACTH), growth hormone (GH) and prolactin did not increase 32P uptake. 3. 3. Treatment with 1 mg of the enzyme-treated pituitary material resulted in a 150 per cent increase in 32P uptake. This response was attributed solely to LH. 4. 4. An assay based on bovine LH standards (NIH-LH-B5) demonstrated that the LH activity of chicken anterior pituitary glands could be measured within the limits of 1·0 to 8·0 gmg.

EFFECT OF THYROXINE ON THE GONADOTROPHIN DOSE RESPONSE LINE USING THE CHICK'S TESTICULAR RESPONSE.

Factorial experiments were carried out to test the interactions between DL-thyroxine and gonadotrophin injections on the gonadal response of cockerels. Mild hyperthyroidism (4 μg DL-thyroxine/day) decreased the index of precision (λ) of the dose response line obtained after subcutaneously injected PMS, FSH and LH, using the increase in testicular weight for PMS and the increase in 32P uptake for FSH and LH. The slope of the dose response line was not affected. The same degree of hyperthyroidism did not decrease the λ of the dose response line obtained after PMS was injected intravenously when 32P was used to measure the response. Administration of 2 μg DL-thyroxine twice a day appeared to increase the 32P uptake by the testes. Higher doses of DL-thyroxine did not yield consistent effects from experiment to experiment. (Endocrinology 76: 194, 1965)