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E F Grollman

Publications and source records attributed to E F Grollman.

At least 37 records · Page 2Linked to original sources
PubMed1986

Characteristics of a lysosomal membrane transport system for tyrosine and other neutral amino acids in rat thyroid cells.

Tyrosine countertransport was used to demonstrate the existence of a carrier system for neutral amino acids in the lysosomal membrane of FRTL-5 thyroid cells. In addition to tyrosine, the carrier system recognized the neutral amino acids leucine, histidine, phenylalanine, and tryptophan. Cystine and lysine, amino acids for which a lysosomal carrier system has been demonstrated, showed no competition with tyrosine for countertransport. The tyrosine system showed stereospecificity and cation independence. It did not require an acidic lysosome or the availability of free thiols. The apparent Km for tyrosine was approximately 100 microM; the energy of activation of the system was approximately 9.7 kcal/mol. This new lysosomal membrane carrier system for neutral amino acids resembles the plasma membrane L system in 3T3 Chinese hamster ovary cells and melanoma B-16 cells.

Amino Acids↗
PubMed1986

Thyrotropin and norepinephrine stimulate the metabolism of phosphoinositides in FRTL-5 thyroid cells.

Hormone-induced changes in phospholipid metabolism were examined in a functioning rat thyroid cell line (FRTL-5). Stimulation of FRTL-5 cells, prelabeled with 32P, with TSH or NE resulted in a rapid decrease in the radioactivity of both phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 4-monophosphate (PIP). The effects of TSH on phospholipid metabolism and calcium mobilization are independent of those on adenylate cyclase. This suggests that the TSH receptor may be unique in that it activates enzyme cascades involved in cAMP production and Ca2+ mobilization.

Animals↗
PubMed1986

Iodine suppression of iodide uptake in FRTL-5 thyroid cells.

Exposure of FRTL-5 cells to iodide (I-) in excess of 3 microM suppresses the concentrative uptake of I-. The depression of I- uptake measured at the steady state is due to decrease in the rate of I- influx and not to an effect on I- efflux. Exposure to NaI is associated with decreased T4 secretion and also depressed Na+-dependent amino acid accumulation. The depression in I- and amino acid transports increases proportionately with the duration of exposure and concentration of I- used but is not associated with alterations in FRTL-5 cell cAMP levels. The I- suppression effect is blocked, however, when methimazole is present during the incubation with NaI. In agreement with studies in vivo, I- suppression in FRTL-5 cells appears to depend on an intermediate in the organification process and to be independent of a TSH-induced cAMP-mediated action.

Amino Acids↗
PubMed1986

Norepinephrine and thyroid-stimulating hormone induce inositol phosphate accumulation in FRTL-5 cells.

3H-Labeled inositol phosphate accumulation is observed when prelabeled FRTL-5 cells (a rat thyroid cell line) are exposed to norepinephrine (NE) or TSH. The presence of inositol trisphosphate among the products implicates a phosphodiesterase-catalyzed hydrolysis of phosphatidylinositol 4,5-bisphosphate. The response to NE is much greater than that to TSH. This may be explained by the ability of cAMP to inhibit inositol phosphate accumulation in these cells. The stimulation by NE is inhibited by alpha 1-adrenergic receptor antagonists and is markedly potentiated in medium of reduced Ca2+ concentration. After chronic withdrawal of TSH from the growth medium, the magnitude of the response to NE is considerably reduced; however, there is no substantial shift in the dose-response curve. This reflects the dependency of alpha 1-adrenergic receptor expression on TSH in the FRTL-5 cell. In contrast, the characteristics of inositol phosphate accumulation induced by acute treatment with TSH are similar in cells maintained in the presence or absence of a low concentration of this hormone, and correlate well with the iodide efflux and iodination of thyroglobulin observed in response to TSH. These results support the hypothesis that TSH may mediate certain of its physiological effects through cAMP-independent mechanisms, such as phospholipid/Ca2+ and C-kinase pathways.

Animals↗
PubMed1984

Gangliosides, the thyrotropin receptor, and autoimmune thyroid disease.

The thyrotropin (TSH) receptor has been proposed to be composed of a membrane glycoprotein and a membrane ganglioside, the former important in high affinity recognition, the latter vital for message coupling to the adenylate cyclase system. The present study used two approaches, formation of antireceptor monoclonal antibodies and reconstitution, to validate the model and further examine the role of the ganglioside. Three kinds of monoclonal antireceptor antibodies are defined. One group which inhibits TSH binding and TSH functions, i.e., TSH-stimulated adenylate cyclase activity, iodide uptake, and thyroid hormone release, is shown to be directed against the glycoprotein component of the receptor. The second group includes antibodies which mimic TSH in all stimulatory actions, are competitive agonists of TSH, are equivalent to thyroid stimulating antibodies in the sera of patients with Graves' disease, and are directed against the ganglioside component of the receptor. These stimulating monoclonal antibodies are directed against a minor ganglioside membrane component which fractionates as a disialoganglioside. When this ganglioside is incorporated into 1-8 thyroid cells which have a correlated ganglioside deficiency and TSH receptor defect, reconstitution of TSH stimulated adenylate cyclase activity occurs. Whereas the first group of antibodies inhibits TSH-stimulated function, they do not inhibit the stimulatory antibodies which mimic TSH, an observation consistent with the 2 component hypothesis of the receptor model. The third group of antibodies have a mix of properties from the first two groups and suggests that the TSH receptor in situ is an actual complex of the two components or that there are common carbohydrate determinants in the functional sites of each receptor component. Implications of a TSH receptor structure in which its ganglioside and glycoprotein components are in equilibrium with pools of free components and, in turn, components important for cholera toxin, tetanus toxin and interferon receptors are discussed. In regard to the pathogenesis of Graves' disease, the data indicate that thyroid stimulating autoantibodies are autoimmune equivalents of cholera toxin with respect to the importance of ganglioside function. Since antiidiotype studies of antibodies against TSH confirm a structural relationship between receptors for thyrotropin, cholera toxin, and thyroid stimulating autoantibodies, the data establish an unequivocal role for the ganglioside in TSH receptor structure which facilitates interpretation of in vitro experiments aimed at understanding the mechanism of ganglioside-ligand interactions.

Animals↗
PubMed1984

Iodide transport in a continuous line of cultured cells from rat thyroid.

The properties of TSH-dependent iodide (I-) uptake are defined for a cloned, continuously growing, functioning rat thyroid cell line (FRTL-5 cells). Since these cells grow without a lumen and are therefore restricted in their ability to iodinate thyroglobulin, the FRTL-5 cells offer the opportunity to directly study transport processes across the membrane into the cell as well as the process whereby I- moves from the cell. FRTL-5 cells concentrate I- approximately 30-fold and exhibit many of the properties of I- uptake seen in thyroid tissue slice and primary cell culture systems. In these cells, accumulation of I- is consistent with a Na+-dependent carrier model for I- uptake, and effects on the influx and efflux processes can be dissociated. Because FRTL-5 cells can be maintained in culture indefinitely and can provide large quantities of a homogeneous functional thyroid cell preparation for study, these cells offer the unique opportunity to further define the mechanism and kinetics of I- transport in a less complex system.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1984

Thyrotropin-stimulated iodide transport mediated by adenosine 3',5'-monophosphate and dependent on protein synthesis.

Iodide (I-) uptake by FRTL-5 cells, a functioning rat thyroid cell line, is TSH dependent. The effects of TSH withdrawal are not apparent until 1 day; 1 week is required to reduce I- uptake to a minimal level. The readdition of TSH leads to a return of the I--concentrating ability after a latency of 12-24 h. The reappearance of I- uptake induced by TSH is mimicked by (Bu)2cAMP and agents that elevate intracellular cAMP levels in these cells, such as forskolin, cholera toxin, and a Graves' disease serum. The appearance of I- uptake after TSH occurs 12 h after the appearance of TSH-induced [35S]methionine incorporation. Cycloheximide blocks both the TSH- and (Bu)2cAMP-induced increases in methionine incorporation and I- uptake to the same extent and in an identical concentration-dependent manner. TSH-induced [35S]methionine incorporation is associated with increased radiolabeling of several specific proteins, as revealed by gel electrophoresis; none, however, is radiolabeled coincident in time with the appearance of TSH-induced I- uptake. Several proteins whose apparent synthesis is induced by TSH also exhibit TSH-dependent phosphorylation.

Animals↗
PubMed1984

Effect of thyrotropin on iodide efflux in FRTL-5 cells mediated by Ca2+.

A functioning rat thyroid cell line (FRTL-5) responds acutely to the addition of TSH, norepinephrine, and the Ca2+ ionophore A23187 by a depression in iodide (I-) uptake levels. The decrease in I- content measured at the steady state depends on the presence of external Ca2+ and can be accounted for by an effect on stimulated I- efflux. As contrasted to the prolonged time (hours and days) involved in the stimulatory effect of TSH on I- uptake, the acute response to TSH is 1) seen within 5 min and maintained for about 20 min, 2) maximum, at a 1 X 10(-7)M concentration of TSH compared with the concentration of 1 X 10(-9)M necessary for the stimulatory effect, 3) independent of whether the cells are growing in the presence or absence of TSH, and 4) not mimicked by the addition of (Bu)2cAMP. The results suggest that TSH and adrenergic stimulation lead to increased membrane permeability to I- which is mediated by an elevation in the intracellular Ca2+ concentration.

Animals↗
PubMed1984

Effect of actinomycin D on iodide transport in FRTL-5 thyroid cells.

TSH-induced I- uptake in FRTL-5 thyroid cells requires new protein synthesis. During the induction of I- uptake, which takes nearly 60 h to reach its maximum, two waves of protein synthesis can be identified: one during the first 8 h and another after 24-30 h, each involving different proteins. Cycloheximide (CH) added during the first 10 h of a 48-h incubation with TSH completely inhibits the induction of I- uptake; 58% inhibition is observed with CH added at 24 h; no inhibition is observed when CH is added 36 h after TSH. Like CH, actinomycin D (ActD), added at the beginning of the 48-h period, inhibits TSH induction of I- uptake; partial inhibition (83%, 72%, and 28%) is observed when ActD is added at 1, 5, and 10 h, respectively. Treatment with ActD at 24 h (ActD-treated cells), however, paradoxically increases I- uptake (1.8- to 3.5-fold over the control value). The characteristics of I- uptake in ActD-treated cells are the same as those in untreated cells; both are Na+ dependent and can be inhibited in a comparable manner by anions. Kinetic measurements of I- transport indicate that ActD increases the rate of I- influx (2-fold or greater increase in maximum velocity without a significant change in Km), with only minor changes in I- release. Enhanced I- uptake in ActD-treated cells is inhibited by the simultaneous (24-h) administration of CH, indicating that protein synthesis is required for the late ActD effect. Despite an overall 2-fold decrease in protein synthesis in cells treated with ActD at 24 h, the synthesis of individual proteins maximally induced by TSH during the first 8 h is increased, whereas that of some proteins maximally synthesized after 24-30 h is markedly reduced. The present data indicate that TSH-induced I- uptake in FRTL-5 cells involves a regulatory action of TSH that operates at the mRNA level.

Animals↗
PubMed1983

Antibodies that promote thyroid growth. A distinct population of thyroid-stimulating autoantibodies.

We used a strain of differentiated rat-thyroid cells in continuous culture (the FRTL-5 strain) to detect the presence of growth-promoting antibodies in serum samples from patients with autoimmune thyroid disease. We found that IgG preparations from 17 of 20 patients (85 per cent) with active Graves' disease and two of five patients (40 per cent) with Hashimoto's thyroiditis could augment thyroid-cell growth. In parallel with IgG-induced elevations in intracellular cyclic AMP levels in the same cell line, all 20 of the patients with active Graves' disease had thyroid-stimulatory antibodies. Patients' IgG preparations fell into three subclasses: those with both potent cyclic AMP stimulation and potent growth-promoting activity; those with potent cyclic AMP stimulation but low-level growth promotion; and those with potent growth promotion and low-level cyclic AMP action. Growth-promoting antibodies were not detected in patients with Graves' disease in remission (seven patients), nodular goiter (seven), subacute thyroiditis (five), or atrophic thyroiditis (one). Simultaneous assays of growth promotion and cyclic AMP stimulation may be useful in the care of patients with autoimmune thyroid disease.

Adult↗
PubMed1983

Ganglioside dependent return of TSH receptor function in a rat thyroid tumor with a TSH receptor defect.

The 1-8 rat thyroid tumor line with a thyrotropin and cholera toxin receptor defect and a deficiency in higher order membrane gangliosides is shown to regain both receptor functions with the in vivo resynthesis or the in vitro reconstitution of higher order gangliosides. Reconstitution was achieved by exposing primary cell cultures of the tumor to preparations of gangliosides from thyroid cells with functional thyrotropin receptor activity.

Adenylyl Cyclases↗
PubMed1983

Graves' IgG stimulation of iodide uptake in FRTL-5 rat thyroid cells: a clinical assay complementing FRTL-5 assays measuring adenylate cyclase and growth-stimulating antibodies in autoimmune thyroid disease.

With optimal conditions and cells maintained in the absence of thyrotropin (TSH) for 7-10 days, IgG preparations from approximately 90% of patients with active Graves' disease can exhibit statistically significant stimulation of cAMP levels in rat FRTL-5 thyroid cells as compared to normal controls. FRTL-5 cells maintained in the absence of TSH for 7-10 days lose their ability to take up iodide. Iodide uptake returns upon readdition of TSH over a 60-hour period via a cAMP-mediated process; thus TSH can be replaced by dibutyryl cAMP or other agents which increase cAMP levels, for example, thyroid-stimulating autoantibodies (TSAbs) from Graves' sera. TSAb stimulation of iodide uptake requires the continued presence of TSAb over at least the first 24 hours of a 48-hour reversal period; TSH, in contrast, can be withdrawn after 5 hours and will still achieve maximal effects at 36-48 hours. Iodide uptake, measured as a 30-minute pulse at 48 hours, appears, however, to be faster with TSAb than TSH. With optimized conditions (cells depleted of TSH greater than 7-10 days; 3-isobytyl-1-methyl xanthine, 0.005 mM; TSAb addition for the entire 48-hour assay period; and a 30-minute pulse of 10 microM 125I-sodium iodide at 37 C), TSAb stimulation is concentration-dependent with a half-maximal activity at approximately 10-fold lower concentrations than in the cAMP stimulation assay. In a series of 24 patients with Graves' disease, IgGs with positive values in the cAMP assay were positive in the iodide uptake assay.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗
PubMed1983

Characterization of the optimal stimulatory effects of graves' monoclonal and serum immunoglobulin G on adenosine 3',5'-monophosphate production in fRTL-5 thyroid cells: a potential clinical assay.

Immunoglobulin G (IgG) preparations derived from the sera of patients with hyperthyroidism due to Graves' disease (TSAb) as well as a monoclonal IgG derived from heterohybridoma fusions of Graves' lymphocytes augmented cAMP levels in a continuous strain of functioning rat thyroid cells (clone FRTL-5) in culture. Optimal stimulation was the same for both types of IgG preparations when measured after 2 h of incubation with 5 X 10(4) cells/well and using cells maintained in a nongrowth, TSH-deficient medium for 7 days. At low IgG concentrations, the stimulatory activities of both preparations exhibited a linear dependence on concentration and similar Ka values (approximately 4 X 10(-8) M) despite the fact that 65% of the Graves' serum IgG preparations had a significantly better ability to inhibit TSH binding to membrane preparations. The Ka value for TSH in the same assay was about 5 X 10(-12) M. Using this cell assay, 90% of a series of hyperthyroid Graves' IgG preparations exhibited stimulating activity, a value comparable to the frequency of positive results found by ourselves and others using human thyroid cell and slice systems. In contrast, only 10% of patients who were euthyroid 1 yr after antithyroid drug withdrawal (n = 21) exhibited stimulating activity, and no stimulating activity was detected in patients with nontoxic nodular goiter (n = 11), toxic adenoma (n = 5), or thyroid carcinoma (n = 6). The studies suggest that an optimized rat FRTL-5 thyroid cell system is a clinically useful and convenient alternative to human thyroid cell and slice systems for detecting TSAbs.

Animals↗
PubMed1982

Monoclonal antibodies to the thyrotropin receptor: stimulating and blocking antibodies derived from the lymphocytes of patients with Graves disease.

Human monoclonal antibodies have been generated from heterohybridomas obtained by fusing mouse myeloma cells with peripheral lymphocytes from patients with active Graves disease. This report characterizes four antibodies as presumptive thyrotropin receptor antibodies because they specifically inhibit thyrotropin binding and competitively inhibit thyrotropin-induced cAMP levels in human thyroid cells. Two of these antibodies, 208F7 and 206H3, are representative of autoimmune stimulators in Graves disease sera because they stimulate thyroid function in all assays, including the mouse bioassay; their ability to inhibit thyrotropin-induced cAMP increases in thyroid cells competitively is complemented by more than additive agonism at low (10 pM) thyrotropin concentrations. These stimulating antibodies interact more potently with human thyroid ganglioside preparations than with bovine thyroid or brain gangliosides; in contrast, they are poor inhibitors of 125I-labeled thyrotropin binding to liposomes containing the glycoprotein component of the human thyrotropin receptor. Antibodies 129H8 and 122G3 appear to be representative of inhibiting or "blocking" antibodies in Graves disease sera. Thus they have no intrinsic stimulatory action in assays of thyroid function but rather inhibit thyrotropin activity in the assays tested. These two antibodies do not react with human thyroid gangliosides but are strong inhibitors of thyrotropin binding to liposomes containing the high-affinity glycoprotein component from human, bovine, and rat thyroid membranes. The data unequivocally establish the pluritopic nature of the immunoglobulins in Graves disease and relate individual components or determinants of the thyrotropin receptor structure with specific autoimmune immunoglobulins.

Antibodies, Monoclonal↗
PubMed1982

Molecular interactions at the cell surface: role of glycoconjugates and membrane lipids in receptor recognition processes.

The present report has described studies using a particular receptor--the TSH receptor--to raise questions concerned with the role of glycolipids and glycoproteins in receptor recognition events and the relevance of lipid modulation of these components in regard to their functional expression. The importance of carbohydrates in other recognition systems will be even more eloquently detailed in subsequent presentations. It is clear, however, that we have gone beyond their recognition as important and are entered into a research stage where questions of how and why are center stage. It is equally clear, as evidenced by the monoclonal antibody data presented herein, that studies of these questions will provide new insight into the mechanism of disease states and will offer new and better diagnostic and therapeutic approaches.

Gangliosides↗
PubMed1981

Calcium transport mechanisms in membrane vesicles from guinea pig brain synaptosomes.

Ca2+ transport mechanisms were investigated using membrane vesicles prepared from guinea pig brain synaptosomes by hypotonic lysis. Two major mechanisms of Ca2+ transport exist, Na+-Ca2+ exchange and ATP-dependent Ca2+ uptake. A third although minor component of Ca2+ uptake occurs under hyperpolarizing conditions (determined by increased uptake of [3H]tetraphenylphosphonium+). Na+-Ca2+ exchange results in a rapid increase of [Ca2+]i (up to 100-fold above [Ca2+]O), has a Km for Ca2+ of 40 microM, is fully reversed by added external Na+, is inhibited by agents dissipating Na+ gradients (monensin or veratridine), and is uninfluenced by mitochondrial inhibitors. ATP-dependent Ca2+ uptake has a higher affinity for CA2+ (Km = 12 microM), is dependent on Mg2+ or Mn2+, and is inhibited by beta, gamma-imidoadenosine 5'-triphosphate and VO43-, although only slightly (20%) inhibited by high concentrations of mitochondrial inhibitors. Both mechanisms are temperature-dependent, fully reversed by A23187, and higher in the presence of external K+. Ca2+ loaded in vesicles via ATP-dependent Ca2+ uptake is rapidly effluxed upon addition of external Na+ (as for Na+-Ca2+ exchange). Therefore a single population of vesicles exists containing both Ca2+ transport mechanisms. The two mechanisms are independent since they accumulate Ca2+ additively, are selectively inhibited by monensin and VO43-, and show distinct specificity toward other divalent cations and La3+. Although independent, Na+ (100 mM) inhibits ATP-dependent Ca2+ uptake (Km for ATP increased from 40 to 300 microM) in the absence of any net Na+ movement. Since Na+-Ca2+ exchange functions in the synaptosomal plasma membrane, the results suggest that both Ca2+ transport mechanisms originate from this membrane and function in the present experiments in inverted plasma membrane vesicles.

Animals↗