Retro-orbital Tissue Research Articles

Thyrotropin-ganglioside interactions and their relationship to the structure and function of thyrotropin receptors.

Gangliosides inhibit 125I-labeled thyrotropin binding to the thyrotropin receptors on bovine thyroid plasma membranes, on guinea pig retro-orbital tissue plasma membranes, and on human adipocyte membranes. This inhibition by gangliosides is critically altered by the number and location of the sialic acid residues within the ganglioside structure, the efficacy of inhibition having the following order: GD1b greater than GT1 greater than GM1 greater than GM2 = GM3 greater than GD1a. The inhibition results from the interaction of thyrotropin and gangliosides, rather than the interaction of membrane and gangliosides. Fluorescence studies show that the inhibition is associated with a distinct conformational change of the thyrotropin molecule and that the progression from a "noninhibitory conformation" to an "inhibitory conformation" parallels exactly the order of effectiveness in inhibiting 125I-labeled thyrotropin binding. The ganglioside inhibition of 125I-labeled thyrotropin binding appears to be hormonally specific in that it is not affected by albumin, glucagon, insulin, prolactin, follicle-stimulating hormone, growth hormone, or corticotropin. The possibility that a ganglioside or ganglioside-like structure is a component of the thyrotropin receptor is suggested by the finding that gangliosides more complex than N-acetylneuraminylgalactosylglucosylceramide are present in bovine thyroid membranes in much higher quantities than have been previously found in extraneural tissue. The finding that the B component of cholera toxin, which also interacts with gangliosides, has a peptide sequence in common with the beta subunit of thyrotropin, suggests that thyrotropin and cholera toxin may be analogous in their mode of action on the membrane.

Stimulation of adenylate cyclase activity in retro-orbital tissue membranes by thyrotropin and an exophthalmogenic factor derived from thyrotropin

Retro-orbital tissue membranes have been shown to have adenylate cyclase activity which can be stimulated by thyrotropin and by an exophthalmogenic factor derived from the thyrotropin molecule by partial pepsin digestion. This stimulable activity is maximal after 15 min and is optimal in the presence of 3 mM magnesium and 1.5 mM ATP. Calcium salts are exquisitely inhibitory to the hormonal stimulation; sodium, lithium, and ammonium salts are significantly less inhibitory. Thyrotropin and the exophthalmogenic factor induce similar maximal levels of stimulation but a 4- to 5-fold higher concentration of exophthalmogenic factor is required to achieve this level. Fluoride stimulates adenylate cyclase activity 2- to 3-fold higher than either thyrotropin or the exophthalmogenic factor; thyrotropin, luteinizing hormone, the beta subunit of thyrotropin, and the alpha subunit of thyrotropin have relative activities for stimulation of cyclase activity of 100:2:2 less than 0.5. Several other polypeptide and glycoprotein hormones have no effect. The gamma-globulin from patients with malignant exophthalmos has no significant effect on cyclase activity either alone or in the presence of maximal levels of thyrotropin or the exophthalmogenic factor; this gamma-globulin does, however, stimulate cyclase activity at submaximal hormone levels. Trypsin not only destroys the hormone-stimulable adenylate cyclase activity on retro-orbital tissue plasma membranes, but also destroys it on the 15,000 to 30,000 molecular weight receptor fragment released from the membranes by the tryptic action.

Experimental exophthalmos. Binding of thyrotropin and an exophthalmogenic factor derived from thyrotropin to retro-orbital tissue plasma membranes

Biologically active preparations of 125I-thyrotropin, [3H]thyrotropin, and the [3H]exophthalmogenic factor derived from thyrotropin by partial pepsin digestion have been used to study the binding properties of the thyrotropin receptor on guinea pig retro-orbital tissue plasma membranes. In regard to the optimal conditions of binding, pH, buffer, salt concentrations, and temperature, these properties are the same as those described in any accompanying report concerning thyrotropin binding to bovine thyroid plasma membranes (Tate, R.L., Schwartz, H.I., Holmes, J.M., Kohn, L.D., and Winand, R.J. (1975) J. Biol. Chem. 250, 6509-6515). In addition, thyrotropin receptors on the retro-orbital tissue plasma membranes are similar to thyrotropin receptors on bovine thyroid plasma membranes in their apparent negative cooperativity and in their relative affinities for luteinizing hormone, the beta subunit of thyrotropin, and the alpha subunit of thyrotropin. In contrast, gamma-globulin from patients with malignant exophthalmos enhances binding when added to incubation mixtures containing the retro-orbital tissue plasma membranes but not when added to those containing thyroid plasma membranes. Normal gamma-globulin and gamma-globulin from Graves' disease patients without exophthalmos do not have this property. The gamma-globulin itself does not bind to the membrane except in the presence of thyrotropin or its exophthalmogenic factor derivative. Tryptic digestion of the retro-orbital tissue membranes releases specific thyrotropin and exophthalmogenic factor binding activity into the supernatant phase. Chromatography on Sephadex G-100 indicates that this trypsin-released receptor activity has a molecular weight of 75,000 or greater, rather than 15,000 to 30,000 for the trypsin-released receptor activity from bovine thyroid membranes (Tate, R.L., Schwartz, H.I., Holmes, J.M., Kohn, L.D., and Winand, R.J. (1975) J. Biol. Chem. 250, 6509-6515).

Structure of an exophthalmos-producing factor derived from thyrotropin by partial pepsin digestion

Previously reported experiments (Winand, R.J., and Kohn, L.D. (1970) J. Biol. Chem. 245, 967-975; Kohn, L.D., and Winand, R.J. (1971) J. Biol. Chem 246, 6570-6575) have demonstrated that partial pepsin digestion of bovine thyrotropin preparation yields a fragment of the thyrotropin molecule which is exophthalmogenic but has negligible or no thyroid-stimulating activity. In the present report this exophthalmogenic derivative of the thyrotropin molecule is shown to contain two major polypeptide components with approximate molecular weights of 14,000 and 6,000. Amino acid analyses, carbohydrate analyses, and tryptic digestion experiments indicate that this exophthalmogenic factor is composed of an intact or nearly intact beta subunit of thyrotropin and an NH2-terminal fragment of the alpha subunit of thyrotropin. Neither polypeptide component of the exophthalmogenic factor has the in vivo exophthalmogenic activity of the intact structure. In vitro the intact exophthalmogenic derivative of the thyrotropin molecule can bind to the thyrotropin receptor on thyroid membranes less efficiently than thyrotropin but significantly better than either its own polypeptide components or the alpha or beta subunits of thyrotropin. The exophthalmogenic factor and its parent thyrotropin molecule can stimulate adenylate cyclase activity in retro-orbital tissue membranes from guinea pigs, a mammalian model of exophthalmos; its polypeptide components have little or no such activity.

Autoimmunity in the pathogenesis of endocrine exophthalmos

It is now fully acceptable that thyroid stimulating antibodies (TSAb) include long-acting thyroid stimulator (LATS) which stimulates the thyroid and adrenal glands in the mouse (El Kabir et al, 1971) and other animals but may have no effect on the human glands, and LATS-protector (LATS-P) or human-specific thyroid-stimulating antibody (HTS) which is probably responsible for the hyperactivity of the thyroid in Graves' disease (Adams, Kennedy and Stewart, 1974; Adams et al, 1974). As in other autoimmune systems there is a larger group of closely allied antibodies, reactive with TSHsensitive receptors in other tissues. Particularly important in this respect are the ophthalmopathic immunoglobulins (O-Ig) which cross-react with the harderian gland in guinea pigs and with adipose tissue cells in several species including man. Such antibodies are cell-stimulating in character as opposed to being cytotoxic, as are thyroid microsomal antibodies. They could be responsible for the proliferation of retro-orbital tissue in exophthalmos; related but not identical antibodies might cause subcutaneous lesions in pretibial myxoedema. There are indications that fibroblasts also proliferate in the orbit and other sites in these conditions but it is uncertain that TSHreceptors exist on connective tissue cells so their stimulation could be due to other mechanisms related to inflammation and repair. Two separate lesions in endocrine exophthalmos are the myositis of the extraocutar muscles and the increased bulk of retrobulbar fat and/or connective tissue. Together these lesions give rise to the diplopia, raised orbital pressure, oedema and proptosis characteristic of the syndrome (Werner, 1972). Autoimmune mechanisms of a different nature are probably involved in the two types of dysfunction. Other factors such as venous stasis play a part in the clinical picture but will not be discussed. The spasm of the levator palpebrae muscle which causes lid retraction and lid lag, is still attributed to the effects of excess circulating thyroid hormones and adrenergic potentiation, and has not so far been implicated immunologically.

The binding of ( 3 H)thyrotropin and an 3 H-labeled exophthalmogenic factor by plasma membranes of retro-orbital tissue.

Plasma membranes of cells from retroorbital tissue have been prepared from the Harderian glands of guinea pigs and have been characterized as being reasonably free of other subcellular structures by electron microscopy and by enzyme-marker analyses. Both bovine thyrotropin and a proteolytic derivative of bovine thyrotropin with exophthalmogenic activity but without thyroid-stimulating activity specifically bind to these membranes. Gammaglobulin from the sera of patients with malignant exophthalmos increases the binding of both pituitary factors, whereas binding is not similarly increased by gammaglobulin from the sera of individuals who are not exophthalmic. The increased binding caused by the gammaglobulin from exophthalmic patients is the same whether the sera are positive or negative for the long-acting thyroid stimulator. Present binding experiments do not indicate a direct interaction between the gammaglobulin and the plasma membranes of the cells from Harderian glands. A mechanism for the pathogenesis of human exophthalmos is proposed on the basis of these data.

Failure of retro-orbital tissue to neutralize the biological activity of the long-acting thyroid stimulator.

Failure of retro-orbital tissue to neutralize the biological activity of the long-acting thyroid stimulator.

The intracellular localization of pituitary thyrotropic hormone.

The intracellular localization of a bovine anterior pituitary preparation of thyroid-stimulating hormone (TSH) was studied in guinea pigs and dogs. The preparation was administered intravascularly or applied directly to tissue sections. TSH was detected by an indirect technique utilizing bovine TSH antiserum and fluorescein-labeled anti-rabbit globulin; the presence of TSH in the tissue was indicated by fluorescence when the tissue was examined under the microscope with an ultraviolet light source. After either intravascular administration or direct application of the TSH preparation, striking fluorescence was found in the nuclei of the thyroid cells and to a lesser degree in the nuclei of retro-orbital fat tissue and kidney tubules in both species studied. A little fluorescence was also seen in spleen tissue. No fluorescence was noted in comparable tissues removed from control animals injected with bovine albumin or globulin or when the tissues were treated with the fluorescein-labeled globulin alone. Fluorescence was also noted in the nuclei of adrenal cells treated with unabsorbed antiserum, but this was greatly diminished when antiserum absorbed with crystalline ACTH was used. The positive reactions were all markedly decreased when the tissues were treated with antisera absorbed with the original TSH preparation. Fluorescence was noted in the cytoplasm of pituitary tissue from both treated and control animals, suggesting a cross-reaction between the bovine pituitary antisera and guinea pig or dog hypophysis. The indirect technique seems to be highly satisfactory for demonstration of the pitiutary hormone within the cell. In addition, the demonstration of immunologically active anterior pituitary TSH bound to cell nuclei offers a clue to the site of action of this hormone.

Sarcoidosis with bilateral exophthalmos as the initial symptom

Bilateral exophthalmos is reported as the initial symptom in a patient with sarcoidosis. The clinical picture and response to therapy were compatible with sarcoidosis and, together with the biopsy data, suggest strongly that the exophthalmos was caused by the presence of sarcoid granulomas in the retro-orbital tissues.

The Retro-Orbital Tissues as a Site of Outflow of Cerebrospinal Fluid

The Retro-Orbital Tissues as a Site of Outflow of Cerebrospinal Fluid