A comparison of lactogenic receptors from rat liver and Nb2 rat lymphoma cells by using cross-linking techniques.

Lactogenic receptors were analysed with the use of the cross-linking agent disuccinimidyl suberate to attach covalently 125I-labelled ovine prolactin or human growth hormone to binding sites from (1) liver from pregnant rats and (2) the rat-derived Nb2 lymphoma cell line. Analysis by SDS/polyacrylamide-gel electrophoresis of the proteins cross-linked to labelled hormone in rat liver indicated a major specifically-labelled complex with an Mr of 68,000-72,000, when run under reducing or non-reducing conditions. With Nb2 cells a major specifically-labelled complex with an Mr of 97,000-110,000 was identified, but only when electrophoresis was run using reducing conditions. Assuming one hormone molecule (Mr 22,000-24,000) per hormone-receptor complex, then the receptor proteins have an Mr of 44,000-50,000 for rat liver and 73,000-88,000 for the Nb2 cells. For both cell types the receptors were of lactogenic specificity; lactogenic hormones competed for binding whereas somatogenic hormones did not. These studies suggest that the lactogenic receptors in rat liver membranes and Nb2 cells differ in two respects. Firstly, the Mr of the labelled receptor protein in Nb2 cells is greater than that of the corresponding receptor protein in rat liver membranes; secondly, the Nb2 cell receptor appears to exist as a disulphide-linked oligomer whereas the receptor in rat liver membranes does not.     

Modulation of thyroid hormone nuclear receptors by cholera toxin in cultured GH1 cells

The cellular actions of the thyroid hormones L-thyroxine and L-triiodothyronine are mediated by the association of hormone with a chromatin-associated receptor. In cultured GH1 cells, a hormone-responsive rat pituitary cell line, thyroid hormone decreases the concentration of its receptor at early incubation times by reducing the accumulation of newly synthesized receptor. In this study, we demonstrate that cholera toxin also reduces the amount of nuclear receptor in GH1 cells in a time- and dose-dependent fashion, without altering the affinity of the receptor for hormone. The reduction of receptor mediated by cholera toxin is not secondary to a generalized inhibition of cell protein synthesis or cell replication rates and this effect can be abolished by pretreatment of the cholera toxin with soluble ganglioside II3-alpha-N- acetylneuraminosylgangliotetraosylceramide . This effect requires an intact cholera toxin molecule and does not occur at similar concentrations of the membrane-binding B subunit of cholera toxin. In order to study the influence of cholera toxin on thyroid hormone receptor turnover, we have used a dense amino acid-labeling technique. The results indicate that cholera toxin does not change the half-life of receptor, but decreases the rate of appearance of newly synthesized receptor. This decreased rate completely accounts for the lowered steady state receptor levels. The extent of cAMP stimulation by cholera toxin does not correlate with the extent of receptor reduction and forskolin, which stimulates cAMP 25- to 500-fold, does not decrease thyroid hormone receptor abundance. These studies suggest that cholera toxin modulates receptor levels by a mechanism(s) that is not mediated by cAMP in GH1 cells.     

Structure and function of cholera toxin and hormone receptors

The enterotoxin from Vibrio cholerae is a protein of 100,000 mol wt which stimulates adenylate cyclase activity ubiquitously. The binding of biologically active ¹²⁵I-labeled choleragen to cell membranes is of extraordinary affinity and specificity. The binding may be restricted to membrane-bound ganglioside G_MI. This ganglioside can be inserted into membranes from exogenous sources, and the increased toxin binding in such cells can be reflected by an increased sensitivity to the biological effects of the toxin. Features of the toxin-activated adenylate cyclase, including conversion of the enzyme to a GTP-sensitive state, and the increased sensitivity of activation by hormones, suggest analogies between the basic mechanism of action of choleragen and the events following binding of hormones to their receptors. The action of the toxin is probably not mediated through intermediary cytoplasmic events, suggesting that its effects are entirely due to processes involving the plasma membrane. The kinetics of activation of adenylate cyclase in erythrocytes from various species as well as in rat adipocytes suggest a direct interaction between toxin and the cyclase enzyme which is difficult to reconcile with catalytic mechanisms of adenylate cyclase activation. Direct evidence for this can be obtained from the comigration of toxin radioactivity with adenylate cyclase activity when toxin-activated membranes are dissolved in detergents and chromatographed on gel filtration columns. Agarose derivatives containing the “active” subunit of the toxin can specifically adsorb adenylate cyclase activity, and specific antibodies against the choleragen can be used for selective immunoprecipitation of adenylate cyclase activity from detergentsolubilized preparations of activated membranes. It is proposed that toxin action involves the initial formation of an inactive toxin-ganglioside complex which subsequently migrates and is somehow transformed into an active species which involves relocation within the two-dimensional structure of the membrane with direct pertubation of adenylate cyclase molecules (virtually irreversibly). These studies suggest new insights into the normal mechanisms by which hormone receptors modify membrane functions.     

Induction of cholera toxin receptors in cultured cells by butyric acid.

Exposure of HeLa cells to sodium butyrate caused an increase in choleragen (cholera toxin) receptors as measured by increased binding of 125I-choleragen to the intact cells. The process was dependent on time and butyrate concentration; maximal increases (over 40-fold) were observed at 48 h and 5 mM sodium butyrate. Other short chain fatty acids were less effective in elevating choleragen receptors in the order: butyrate greater than pentanoate greater than hexanoate greater than propionate. Acetate and isobutyrate had no effect. The increase in toxin receptors caused by butyrate was reversible and occurred in serum-free medium. The affinity of choleragen for control and butyrate-treated HeLa cells appeared to be similar. Butyrate also induced an elevation in choleragen receptors in rat C6 glial and Friend erythroleukemic cells but not in a butyrate-resistant HeLa mutant. The increase observed in Friend cells paralleled the increase in ganglioside GM1 (galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide), the reported choleragen receptor. Although no GM1 could be detected in untreated Hela cells, small amounts were found in cells exposed to butyrate.     

Dissociation by cooling of hormone and cholera toxin activation of adenylate cyclase in intact cells

Cholera toxin, through adenylate cyclase activation reproduced cyclic AMP-mediated effects of thyroid-stimulating hormone (TSH) in dog thyroid slices, i.e protein iodination, [1-¹⁴C]glucose-oxidation and hormone secretion. Iodide and carbamylcholine decreased the cyclic AMP accumulation induced by cholera toxin as well as by TSH, which supports the hypothesis of an action of these agents beyond the steps of hormone-receptor and receptor-adenylate cyclase interaction. Cooling to 20°C did not impair the TSH induced cyclic AMP accumulation in thyroid slices, but completely suppressed the cholera toxin effect.This observation has been extended to other hormones and target tissues, such as the parathyroid hormone (PTH) (kidney cortex), adrenocorticotropic hormone (ACTH) (adrenal cortex)_and luteinizing hormone (LH) (ovary systems). As in thyroid, cooling dissociated the cholera toxin and hormonal effects on cyclic AMP accumulation. In homogenate, cooling decreased cyclic AMP generation in the presence of cholera toxin but at 20°C and 16°C a cholera toxin stimulation was still observed. These results bear strongly against the hypothesis that the glycoprotein hormones TSH and LH activate adenylate cyclase by a mechanism identical to cholera toxin.     

Dissociation by cooling of hormone and cholera toxin activation of adenylate cyclase in intact cells.

Cholera toxin, through adenylate cyclase activation reproduced cyclic AMP-mediated effects of thyroid-stimulating hormone (TSH) in dog thyroid slices, i.e. protein iodination, [1-14C]glucose-oxidation and hormone secretion. Iodide and carbamylcholine decreased the cyclic AMP accumulation induced by cholera toxin as well as by TSH, which supports the hypothesis of an action of these agents beyond the steps of hormone-receptor and receptor-adenylate cyclase interaction. Cooling to 20 degrees C did not impair the TSH induced cyclic AMP accumulation in thyroid slices, but completely suppressed the cholera toxin effect. This observation has been extended to other hormones and target tissues, such as the parathyroid hormone (PTH) (kidney cortex), adrenocorticotropic hormone (ACTH) (adrenal cortex) and luteinizing hormone (LH) (ovary systems). As in thyroid, cooling dissociated the cholera toxin and hormonal effects on cyclic AMP accumulation. In homogenate, cooling decreased cyclic AMP generation in the presence of cholera toxin but at 20 degrees C and 16 degrees C a cholera toxin stimulation was still observed. These results bear strongly against the hypothesis that the glycoprotein hormones TSH and LH acetivate adenylate cyclase by a mechanism identical to cholera toxin.     

Inhibition of the proliferation of Nb2 cells by femtomolar concentrations of cholera toxin and partial reversal of the effect by 12-O-tetradecanoyl-phorbol-13-acetate

One hour of exposure to cholera toxin is sufficient to elicit a significant delay in the initiation of DNA synthesis and cell division in lactogenic hormone-dependent Nb2-11C lymphoma cells. The inhibitory effect occurs already at very low concentrations of cholera toxin (5-50 fM), at which it is not accompanied by a detectable increase in intracellular cAMP, or ADP-ribosylation of the alpha subunit of Gs, the stimulatory guanine nucleotide binding protein of adenylate cyclase; IBMX, the phosphodiesterase inhibitor, acts synergistically to cholera toxin, indicating that a minute increase in cAMP may be sufficient for the inhibition. This indication is substantiated by the finding that dibutyryl cAMP also inhibits cell proliferation. Phorbol diester reverses partially the inhibitory activity of cholera toxin. It is most likely that this effect does not result from blocking the increase in cAMP, but rather from some subsequent, yet unidentified, events. The inhibitory effect of cholera toxin is not dependent on the concentration of the proliferation-stimulating lactogenic hormone and cannot be abolished or reduced by excess of the hormone. Cholera toxin also inhibits the autonomous proliferation of a lactogenic hormone-independent cell line (Nb2-SP); however, in this case the inhibition is not affected by TPA.     

Reduction of adenylyl cyclase activity by cholera toxin in myeloid cells. Long-term down-regulation of Gs alpha subunits by cholera toxin treatment

In IPC-81 cells, the adenylyl-cyclase activation by cholera toxin produces an elevation of cAMP that causes a rapid cytolysis. A resistant clone with deficient cholera toxin-induced cyclase activity (yet sensitive to cAMP) showed a rapid decrease in the amount of membrane-bound Gs alpha (42-47 kDa) detectable soon after ADP-ribosylation of these proteins; pertussis toxin-sensitive G proteins (41 kDa) were not affected. Resistant cells showed a rapid decrease of Gs alpha that is consistent with the finding that cAMP did not accumulate in these cells. Cholera toxin treatment of resistant cells had long-lasting effects (several weeks) on the level of Gs alpha in the cell membrane. The duration of Gs alpha decrease does not correspond to the probable life of catalytically active cholera toxin in the cells, and suggests a regulated process more complex than a proteolytic degradation targeted on ADP-ribosylated molecules.     

Down-regulation of glucocorticoid receptors in mouse lymphoma cell variants.

The mouse thymoma-derived cell line W7 is sensitive to the cytolytic action of glucocorticoids. We have isolated a novel class of cell variant that apparently overcomes its inherent sensitivity to glucocorticoids by reversibly down-regulating the level of glucocorticoid receptors. This phenotype is stable during subcloning in the presence and in the absence of glucocorticoids and is dominant in somatic cell hybrids with wild-type cells. Fusion of this variant with wild-type cells produces hybrids that down-regulate and are less sensitive to glucocorticoids than hybrids of receptor-negative and wild-type cells. This is the first demonstration of a phenotypic change which correlates with down-regulation of the glucocorticoid receptor.     

Histochemical and immunocytochemical localization of prolactin receptors on Nb2 lymphoma cells: applications of confocal microscopy

We studied prolactin (PRL) binding sites on Nb2 lymphoma cells using two different light microscopic methods. First, histochemical detection was accomplished by using an aminomethyl coumarin-acetic acid-conjugated ovine prolactin molecule (AMCA-oPRL) on both glutaraldehyde-fixed and unfixed Nb2 lymphoma cells. Binding of AMCA-oPRL was studied after UV illumination and appeared as punctate fluorescence associated with many but not all cells. Binding was abolished when tissue sections were treated with excess unlabeled lactogenic hormones and was unchanged when a non-lactogenic hormone was used for displacement. Counting revealed significant differences between the number of labeled cells in populations known to exhibit up- or down-regulated PRL receptors. Second, indirect immunocytochemistry of Nb2 PRL receptors was accomplished by immunological detection of exogenously added ovine PRL using two antisera directed against ovine PRL. Visualization of the ligand-antibody complexes was accomplished by confocal laser scanning microscopy. Staining was restricted to a subpopulation of cells. The morphological results presented here add to the previous physiological and biochemical data on the presence of lactogenic hormone receptors on Nb2 lymphoma cells.     

Internalization and degradation of cholera toxin by cultured cells: relationship to toxin action

Using anticholeragen antibodies and 125I-protein A, we developed a specific and quantitative assay for measuring choleragen on the surfaces of cultured cells. When neuroblastoma cells containing bound toxin were incubated at 37 degrees C, surface toxin disappeared with a half-life of approximately 2 h and a significant loss was detected by 10 min. When cells were incubated with 125I-choleragen in order to measure toxin degradation, cell-associated radioactivity disappeared with time and a corresponding amount of TCA-soluble label appeared in the culture medium with a half-life of 4-6 h. No degradation was detected until 45 min. Although there was a lag of 15 min before bound choleragen activated adenylate cyclase, the enzyme became maximally activated between 45 and 60 min. Similar results were obtained with Friend erythroleukemia cells. Internalization, degradation, and activation all were blocked when the cells were maintained at 4 degrees C. At 22 degrees C, internalization and activation occurred, albeit at a slower rate, whereas degradation was effectively inhibited. These results indicated that choleragen does not have to be degraded by intact cells in order for it to activate adenylate cyclase. Some internalization of the toxin, however, appears to precede the activation process.     

Down-regulation of plasmin receptors on human sarcoma cells by glucocorticoids

After incubation of confluent monolayer cultures of human HT-1080 fibrosarcoma cells with purified native human plasminogen in plasminogen-depleted serum-containing medium, bound plasmin activity could be specifically eluted from the cells with tranexamic acid, an analogue of lysine. Dexamethasone reduced the amount of recoverable bound plasmin activity in a dose-dependent manner. Dexamethasone was also found to induce a time- and dose-dependent decrease in the ability of the cells to bind added plasmin. Untreated HT-1080 cells bound added plasmin with a high capacity (600,000 molecules bound per cell), and this decreased to an undetectable level after treatment with 100 nM dexamethasone. The kinetics of the loss of plasmin binding by the dexamethasone-treated sarcoma cells, a clear decrease after 4 h, correlated with those for the loss of cell-bound urokinase (u-PA) activity. Plasmin was not, however, bound to the active site of u-PA: an anti-catalytic monoclonal antibody to u-PA had no effect on plasmin binding. Other glucocorticoids, such as hydrocortisone and corticosterone, had a similar effect to dexamethasone on plasmin binding to HT-1080 cells. The effect of glucocorticoids on the plasmin receptor seemed to occur at least partly via a decrease in the affinity for plasmin, since the Kd for plasmin with untreated cells was 5.4 x 10(-9) M, and with cells treated with 5 nM dexamethasone, the Kd value for plasmin was 1.2 x 10(-7) M. These results show that glucocorticoids induce down-regulation of plasmin receptors on the surface of HT-1080 cells: a novel mechanism, in addition to the known effects of glucocorticoids on u-PA and PA inhibitors, by which human tumor cells may be disarmed of their pericellular proteolytic activity.     

Determination of lactogenic activity in the serum of the squirrel monkey (Saimiri boliviensis) using the Nb2 lymphoma bioassay

Determination of squirrel monkey prolactin by immunoassay has been hampered by the lack of antiserum specific to prolactin from this species. As an alternate method, we have investigated whether the Nb2 lymphoma bioassay could be adapted for routine measurement of the lactogenic activity of samples of squirrel monkey serum. The growth of the Nb2 cells is absolutely dependent on the presence of lactogens in the culture medium. The cells were maintained in Fisher's medium supplemented with 10% horse serum, 10% fetal calf serum (FCS), and 10^?4M β-mercaptoethanol. For each assay, the cells were plated at an initial density of 1 × 10⁵ cells/ml in 22-mm 12-well dishes in the above medium, but devoid of FCS. Serum samples were heated to 56°C for 20 minutes to abolish the unusually high cytolytic complement activity of squirrel monkey serum and were incubated for 72 hours with Nb2 cells at serial dilutions from 1/40 to 1/2,560. Growth curves were generated with pooled samples of squirrel monkey serum, and the level of lactogenic activity was estimated using a calibration growth curve generated with known concentrations of purified rhesus monkey prolactin standard. We have found that the Nb2 lymphoma bioassay provides a sensitive and adaptable means for determination of lactogenic activity in the serum of the squirrel monkey.     

Modulation of human growth hormone binding to somatogenic and lactogenic receptors by monoclonal antibodies to human growth hormone.

The relationship between the structure of human growth hormone (hGH) and the hormone-receptor interaction was investigated by studying the effects of specific monoclonal antibodies (MAbs) to hGH on the binding of [125I]hGH to rabbit liver and mouse liver microsomes. Receptor binding assays were carried out using a constant dose (1 ng) of [125I]hGH and varying concentrations of MAbs. The assay was carried out in the presence of either excess ovine prolactin for the measurement of somatogenic (SOM) binding sites, or excess bovine growth hormone for the determination of lactogenic (LAC) binding sites. Anti-hGH MAbs were found to have a whole spectrum of effects on hGH binding, including inhibitory, non-effect and enhancing activities. Enhancement of the binding of [125I]hGH to both SOM and LAC receptors was observed in liver membranes of rabbit or mouse. The observed amplified signal of [125I]hGH binding to various receptors in the presence of MAb no. 8 may be due to conformational changes which occur following MAb binding to hGH. On the other hand, most of the other MAbs caused inhibition of [125I]hGH binding. A negative correlation exists between the cross-reaction of various MAbs with the N-terminus truncated forms of hGH (Met14-hGH or Met8Leu-hGH) and their respective KD/IC50 values enabled the evaluation of the crucial role of the N-terminus region in hGH binding to both LAC and SOM receptors. MAb nos 1 and 19, which are directed towards acid residues 95-134 and the C-terminus, inhibited SOM binding more potently than LAC binding. Thus, it seems that these mid-molecule and C-terminus regions are also important in hGH binding, and that they play a role in the partial overlap of SOM and LAC binding.     

Butyrate-induced reversal of dexamethasone resistance in autonomous rat Nb2 lymphoma cells

The parental rat Nb2 lymphoma is a prolactin (PRL)-dependent T cell line. Exposure of a PRL-independent subline, Nb2-SFJCD1, to sodium butyrate (NaBT) causes transient reversal of their growth factor-independent proliferation in association with constitutive expression of protooncogenes pim-1and c-myc. In the present study, we investigated the effect of NaBT treatment on the sensitivity of Nb2-SFJCD1 cells to dexamethasone (DEX)-induced apoptosis. Pretreatment with NaBT (2 mM, 72 h) partially reversed resistance to apoptosis in Nb2-SFJCD1 cells exposed to DEX (100 nM) for 12 h, assessed by flow cytometric analyses of DNA fragmentation. However, the cytolytic effect of DEX was abrogated by PRL i n a time- and concentration-dependent manner. Eval uati on of apoptosis-associated gene expression in NaBT-pre-treated cultures incubated with DEX or DEX+PRL indicated that the apoptosis resistance did not stem from altered bcl-2 or bax expression. However, there was a strong correlation between the resistance to DEX-activated apoptosis and their enhanced expression of pim-1 mRNA and protein. The results show that it is possible to reverse DEX-induced apoptosis of Nb2 pre-T cells and suggest the pim-1 gene product has an important role as a suppressor of this process, perhaps functioning as a mediator of PRL action. This revised version was published online in June 2006 with corrections to the Cover Date.     

Metabolic responses of Sertoli cells in culture to various concentrations of follicle stimulating hormone and cholera toxin.

The concentration of cholera toxin required for half-maximal stimulation of cAMP production by Sertoli cell enriched cultures (4.48 X 10(2) microgram/ml) is greater than that required for half-maximal stimulation of 17beta-estradiol synthesis from testosterone (2.34 X 10(-4) microgram/ml), [3H]thymidine incorporation into DNA (1.48 X 10(-5) microgram/ml), or androgen binding protein production (2.43 X 10(-6) microgram/ml). The same relative dose response hierarchy was obtained with respect to stimulation of Sertoli cells with follicle stimulating hormone (FSH) preparations. Again, highest concentrations were required to elicit maximal cAMP production. The data are discussed in relation to an apparent paradox: If cAMP is the mediating 'second messenger' following stimulation by FSH or cholera toxin, why should highest concentrations of these agents be required to elicit 50% of maximal cAMP levels?     

Human growth hormone binds to lactogenic receptors in bovine, ovine and rat adrenals

The distribution of 125I radioactivity in the liver, kidneys, adrenals and serum of male rats was measured 10 minutes after an intravenous bolus of 125I-labelled human growth hormone (hGH) was administered in the presence or absence of a large excess of ovine growth hormone or ovine prolactin. The hGH binding sites in the adrenals had displacement properties characteristic of lactogenic receptors, whereas those in the liver had displacement properties characteristic of somatogenic receptors. Bovine and ovine adrenal microsomal membrane fractions contained high affinity (Ka = 1.4-3.3 nM-1) binding sites for hGH which showed ligand specificity typical of lactogenic receptors. It is concluded that the hGH binding site in the adrenal gland is a classical lactogenic receptor and that this tissue is a convenient and rich (42.6 +/- 6.4 fmol hGH specifically bound/mg protein) source of receptor suitable for further characterization.