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R W Harrison

Publications and source records attributed to R W Harrison.

At least 109 records · Page 6Linked to original sources
PubMed1981

Conditions affecting AtT-20 cell glucocorticoid uptake.

Glucocorticoid uptake by intact AtT-20/D-1 cells was studied to determine if the extent of uptake was enhanced or retarded by binding components in serum. The results demonstrate that the uptake of corticosterone, which binds to transcortin, was reduced by addition of serum while uptake of triamcinolone acetonide, which is not bound by transcortin, was unaffected. Neither heat denatured serum nor bovine serum albumin affected corticosterone uptake, further emphasizing the specificity of the inhibition. The presence of serum also affected the apparent binding specificity, since steroids able to bind to transcortin became less effective competitors when serum was present in the incubation medium. In the absence of serum, the specificity of glucocorticoid uptake was qualitatively similar to that of the isolated cytosol receptor. These results emphasize that the selective inhibitory effect of serum transcortin on whole cell uptake of certain steroids should be considered when assaying steroid potency in intact cellular systems.

Animals↗
PubMed1979

Cell-free synthesis of mouse corticotropin. Evidence for two high molecular weight gene products.

Polysomes or mRNA prepared from cultured AtT-20/D16v mouse pituitary adenocarcinoma cells direct the efficient incorporation of amino acid into newly synthesized material in the presence of wheat germ translational factors. A significant franction of the total cell-free product is specifically immunoprecipitable with corticotropin antibody purified by immune affinity chromatography. Analysis of the cell-free synthesized immunoreactive products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals that two high molecular weight corticotropin species (Mr congruent to 32,500 and 28,000) are synthesized in an approximate 2:1 ratio. Neither product contains carbohydrate based upon concanavalin A chromatography or exposure to polysaccharidases. The smaller molecular weight product does not appear to arise from proteolytic processing since both species are synthesized in approximately the same ratio in cell-free reaction mixtures directed by either polysomes or mRNA. These results suggest that AtT-20/D16v cells contain two distinct mRNA poluations specifying the synthesis of two different high molecular weight forms of mouse corticotropin.

Adenocarcinoma↗
PubMed1979

Corticosterone binding in AtT-20 pituitary tumor cell cytosol: evidence for one class of binding site for both natural and synthetic glucocorticoids.

The AtT-20 mouse pituitary cell is an established, cloned cell line which produced adrenocorticotrophic hormone in a glucocorticoid-suppressible manner. A receptor for glucocorticoids was identified in cytosol prepared from these cells using the natural mouse glucocorticoid, corticosterone, as the labeled ligand. The question of whether this binding component is identical to the one detectable using labeled triamcinolone acetonide was addressed by comparing their physicochemical characteristics and by detailed studied of binding specificity using both ligands. The corticosterone and triamcinolone acetonide binding components behaved similarly on sucrose density gradient analysis and DEAE-cellulose ion-exchange chromatography. Scatchard analysis with corticosterone detected 30% fewer binding sites than a similar analysis with triamcinolone acetonide, probably because corticosterone binding was of lower affinity (Kd = 8.6 . 10(-9)M vs. 1.4 . 10(-9)M) and hence less stable. The relative glucocorticoid binding affinities of thirteen unlabeled steroids were obtained using either labeled steroid as ligand. Both ligands yielded similar results, suggesting that they both detected a similar binding site. The results suggest that AtT-20 cell cytosol contains a single class of binding site which detects both natural and synthetic glucocorticoids.

Animals↗
PubMed1979

Heterogeneity of AtT-20 cell glucocorticoid binding sites: evidence for a membrane receptor.

In previous studies we have found that intact AtT-20 cells contained two glucocorticoid binding sites with distinctly different affinities and specificity. In this paper, the nature of these sites was investigated by studying glucocorticoid binding to cytosol and to plasma membranes isolated from AtT-20 mouse pituitary tumor cells. Plasma membrane vesicles were isolated from AtT-20 cells and found to take up alpha-aminoisobutyric acid, indicating that they were properly oriented and functionally intact. Corticosterone bound to these vesicle in a time- and temperature-dependent manner. The binding exhibited a glucocorticoid preference since non-glucocorticoids such as progesterone, testosterone or estradiol were unable to inhibit binding. In addition, binding specificity differed from that of the cytoplasmic receptor since the synthetic glucocorticoids were also ineffective competitors. The major inhibitors of binding were corticosterone greater than 11-dehydrocorticosterone greater than 11-ketoprogesterone greater than cortisol. In other complementary studies, AtT-20 cell cytosol was tested to determine whether heterogenous soluble sites exhibiting a preference for the natural vs the synthetic steroid could also be identified. We found that binding sites for both steroid classes were approximately similar in number, specificity and behavior on ion-exchange chromatography. We conclude that, in addition to a classical soluble cytoplasmic glucocorticoid receptor, AtT-20 cells contain plasma membrane glucocorticoid binding sites. The affinity and specificity of these sites for the natural ligand, corticosterone, suggest that they play an important role in the subcellular mechanism of glucocorticoid action.

Aminoisobutyric Acids↗
PubMed1979

Evidence for glucocorticoid transport into AtT-20 cells. Selective effect of a sulfhydryl inhibitor on uptake.

The purpose of these experiments was to determine if membrane-mediated glucocorticoid uptake by the AtT-20 cell was sensitive to the sulfhydryl group inhibitor, p-chloromercuriphenylsulfonate (PCMPS). This agent was chosen because of its reported limited entry into the cell interior. Our experimentes showed that cells could be incubated with 1 mM PCMPS for 1 h before intracellular concentrations of the inhibitor were sufficient to affect the intracellular cytosol receptor. In contrast less than 15 min of treatment inhibited intact cell steroid uptake. The inhibition was completely reversed by dithiothreitol. These studies show inhibition of glucocorticoid uptake under conditions which do not affect the intracellular receptor and infer that the AtT-20 cell membrane-mediated uptake mechanism for glucocorticoids contains sensitive sulfhydryl groups.

4-Chloromercuribenzenesulfonate↗
PubMed1979

Uniform depth dose distribution for biological irradiation using negative pions.

A simple, flexible technique has been developed to generate uniform depth dose profiles for the biomedical pion beam at TRIUMF using dynamic momentum control and linear programming. Either the entrance dose or the irradiation time required for a certain dose over the uniform region can be minimised. The dynamic momentum control can operate automatically under computer control even with a highly unstable beam. Cell survival profiles have been obtained for this uniform dose distribution using the gelatin technique. The RBE increases with increasing depth through the uniform dose region.

Animals↗
PubMed1979

The ontogony of the human placental glucocorticoid receptor and inducibility of heat-stable alkaline phosphatase.

The human placenta was found to contain a cytosol receptor for glucocorticoids. The concentration of this receptor in term placenta was 27-fold higher than that found in cytosol from first trimester placenta. The levels of cytosol glucocorticoid receptor in three trophoblastic cell lines (JAr, BeWo, and JEG) were also determined and all were found to be low. The ability of prednisolone, a potent glucocorticoid, to stimulate heat-stable alkaline phosphatase activity found in these cells was tested. Although control experiments demonstrated that the conditions were adequate to stimulate HeLa cell alkaline phosphatase, none of the trophoblastic lines responded to prednisolone administration. This result may be explained by the observation that the JAr cells lacked any detectable glucocorticoid receptor and the receptor levels in cytosol prepared from JEG and BeWo cells were 12% and 2%, respectively, of those measured in HeLa cytosol. Our studies also suggest that the increase in serum levels of heat-stable alkaline phosphatase observed during pregnancy may reflect increasing placental sensitivity to glucocorticoids as a result of increased receptor levels.

Alkaline Phosphatase↗
PubMed1977

The effect of cell membrane alteration on glucocorticoid uptake by the AtT-20/D-1 target cell.

The glucocorticoid-sensitive AtT-20/D-1 cell line was used to study cellular uptake of glucocorticoids. A previous observation that glucocorticoid uptake by these cells was temperature dependent had prompted us to postulate that glucocorticoids entered the cell by a temperature-sensitive transport process located in the cell membrane. Attempts were then made to perturb the membrane mechanism. In some of these experiments, intact cells were treated with neuraminidase or pronase. The release of sialic acid in the case of neuraminidase treatment and of sialic acid and cell surface peptides in the case of pronase treatment demonstrated that the enzymes were effective. Approx. 60% of total cellular sialic acid was released by a 15 min incubation with 20 microng/ml neuraminidase at 25 degrees C. The treated cells appeared to be viable, in that they continued to produce corticotropin at a normal rate, yet intact cell glucocorticoid binding at both 4 and 25 degrees C was only 20-30% of that of untreated cells. Treatment with pronase also caused steroid uptake at 4 and 25 degrees C to be reduced, although the extent of reduction was less than that seen following neuraminidase treatment. In other experiments, the effect of exposure of AtT-20/D-1 cells to ethanol or dimethyl sulfoxide was determined. The solvent concentrations used (0.5-10%) did not alter cell viability significantly, and the ability of the cytosol receptor to bind steroid in a cell-free preparation was unimpaired. However, incubation of intact cells with 10% (v/v) dimethyl sulfoxide or ethanol resulted in an 80-90% decrease in steroid uptake at 25 degrees C. We conclude that steroid uptake by the intact cell can be perturbed by treatments which do not affect the cytosol receptor or alter cell viability. These results support the postulate that glucocorticoids enter the AtT-20/D-1 cell by a specific membrane-associated mechanism.

Animals↗