Evelyn F. Grollman

Monocarboxylate transporter MCT1 is located in the apical membrane and MCT3 in the basal membrane of rat RPE

The retinal pigment epithelium (RPE) forms the outer blood-retinal barrier and regulates the movement of nutrients, water, and ions between the choroidal blood supply and the retina. The transport properties of the RPE maintain retinal adhesion and regulate the pH and osmolarity in the space surrounding the photoreceptor cell outer segments. In this report we identify two monocarboxylate transporters, MCT1 and MCT3, expressed in rat RPE. On the basis of Northern and Western blot analyses, MCT1 is expressed in both the neural retina and the RPE, whereas the expression of MCT3 is restricted to the RPE. Using indirect immunolocalization we show that the two transporters are polarized to distinct membrane domains. MCT1 antibody labels the apical surface and the apical processes of the RPE. A polyclonal antibody produced against the carboxy terminus of rat MCT3 labels only the basolateral membrane of the RPE. The demonstration of MCT1 on the apical membrane and MCT3 on the basal membrane identifies specific proteins involved in the discriminate and critical regulation of water and lactate transport from the retina to the choroid.

An Enzymatic Basis for Lewis Blood Types in Man

Milk from women with blood type Le(a+) or Le(b+) contains a specific fucosyltransferase not found in the milk of women with blood type Le(a- b-). The enzyme, a guanosine diphosphate L-fucose: N-acetyl-beta-D-glucosaminylsaccharide alpha-4-L-fucosyltransferase is apparently required for the synthesis of the structural determinants of Le(a) and Le(b) specificity, both of which contain fucose in an alpha-1,4 linkage to N-acetylglucosamine. The same enzyme is also involved in the synthesis of milk oligosaccharides, as two oligosaccharides which contain this linkage are absent from the milk of women with Le(a- b-) blood type.

Differences in the electrophysiological response to I− and the inhibitory anions SCN∮- and C1O4−, studied in FRTL-5 cells

The electrophysiological properties of the Na+/I- symporter (NIS) were examined in a cloned rat thyroid cell line (FRTL-5) using the whole-cell patch-clamp technique. When the holding potential was between -40 mV and -80 mV, 1 mM NaI and NaSCN induced an immediate inward current which was greater with SCN- than with I-. The reversal potential for I- and SCN- induced membrane currents was +50 mV. This is close to the value of +55 mV calculated by the Nernst equation for Na+. These results are consistent with I- and SCN- translocation via the NIS that is energized by the electrochemical gradient of Na+ and coupled to the transport of two or more Na+. There was no change in the membrane current recording with ClO-4 indicating that ClO-4 was either not transported into the cell, or the translocation was electroneutral. ClO-4 addition, however, did reverse the inward currents induced by I- or SCN-. These effects of I-, SCN- and ClO-4 on membrane currents reflect endogenous NIS activity since the responses duplicated those seen in CHO cells transfected with NIS. There were additional currents elicited by SCN- in FRTL-5 cells under certain conditions. For example at holding potentials of 0 and +30 mV, 1 mM SCN- produced an increasingly greater outward current. This outward current was transient. In addition, when SCN- was washed off the cells a transient inward current was detected. Unlike SCN-, 1-10 mM I- had no observable effect on the membrane current at holding potentials of 0 and +30 mV. The results indicate FRTL-5 cells may have a specific SCN- translocation system in addition to the SCN- translocation by the I- porter. Differences demonstrated in current response may explain some of the complicated influx and efflux properties of I-, SCN- and ClO-4 in thyroid cells.

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‐mono‐phosphate (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.

Monoclonal antibodies to the thyrotropin receptor: the identification of blocking and stimulating antibodies

Monoclonal antibodies to the thyrotropin (TSH) receptor have been obtained from fusions of mouse myeloma cells with spleen cells immunized with solubilized thyroid membrane preparations. Two monoclonal antibodies which inhibit 125l-TSH binding and are reactive with the glycoprotein component of the bovineTSH receptor (11E8 and 13D11), are shown to inhibit basal and TSH stimulated adenylate cyclase activity in bovine thyroid membranes and human thyroid cells. Both antibodies also inhibit 125l-TSH binding in vitro, whether binding is measured at pH 6.0 in low salts and at 0–4 C or at pH 7.4 in 50 mM NaCl and at 37 C. The glycoprotein component is thus a portion of the physiologic TSH receptor in vivo and 125l-TSH binding studies apparently measure the high affinity glycoprotein component under nonphysiologic conditions and conditions more representative of the physiologic milieu. A third monoclonal antibody whose interaction with thyroid membranes is prevented by TSH is shown to stimulate adenylate cyclase activity in bovine thyroid membranes and human thyroid cells. This stimulating antibody only weakly inhibits 125l-TSH binding to thyroid membranes or to the glycoprotein component of the TSH receptor. The 22A6 antibody does, however, immunoprecipitate mixed brain gangliosides, in distinct contrast to the monoclonal antibodies to the glycoprotein receptor component, i.e., 11E8 and 13D11. The results support the speculation that autoimmune antibodies which inhibit TSH binding to thyroid membranes are not necessarily identical to antibodies which stimulate function; that antibodies directed at the high affinity initial site of TSH interaction with a cell can behave as blocking rather than stimulating antibodies and that a possible relationship exists between stimulating antibodies and the low affinity TSH binding sites (gangliosides) on thyroid membranes.

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 ∼ 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 > 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 µM 125I-sodium iodide at 37 C), TSAb stimulation is concentration-dependent with a half-maximal activity at ∼ 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. Activity coefficients expressed as percent of basal in both assays correlate extremely well (r = 0.85). Using these two assays and an FRTL-5 assay measuring growth antibodies (by their ability to increase thymidine uptake), TSAbs were found in 100% of patients with active Graves’ disease and could broadly be grouped into three categories: IgGs with high stimulatory cAMP/iodide uptake activity but low to moderate effects on thymidine uptake; IgGs with moderate effects in all assays; and IgGs with low or undetectable cAMP/iodide stimulatory activity, but high thymidine uptake activity. The value of the FRTL-5 rat thyroid cells assays system for measuring Graves’ autoantibodies appears, thus, to be superior to all current systems, including human cells and slices.

Thyrotropin Receptors and Gangliosides

Recent studies (1, 2, 28–30, 33–35) have implicated ganglio-sides in the receptor recognition process for thyrotropin (TSH) and have led to the hypothesis (15–17, 19) that the TSH receptor is structurally and functionally related to receptors for other ligands which interact with gangliosides in vitro, i.e., cholera toxin, tetanus toxin, and interferon. This report summarizes the accumulated evidence which implicates both a glycoprotein (4, 38–40, 43) and a ganglioside in the structure and function of the TSH receptor. In the context of this symposium, particular attention will be addressed to the role of the ganglioside.

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.

Clinical and morphologic findings of familial goiter in bongo antelope (Tragelaphus eurycerus).

Inherited defects of thyroglobulin synthesis resulting in congenital goiter are well described in certain breeds of domestic ungulates and in human beings. Goiter associated with synthesis of an abnormal thyroglobulin and the presence of thyroidal albumin was identified in five closely related bongo antelopes (Tragelaphus eurycerus). The goiter had an adult onset, and the affected bongos appeared to remain euthyroid with normal serum T3 and T4 values, normal serum cholesterol concentrations, and nonelevated concentrations of circulating thyroid stimulating hormone (TSH). Goitrous bongos had significant reproductive difficulties, including reduced cyclic activity and prolonged gestations, but were otherwise normal. Over the course of the disease, the thyroid glands greatly enlarged (up to 10 × 20 cm) and became polycystic. Microscopically, there was an admixture of giant colloid-filled follicles and follicles of normal size lined with variable follicular epithelium ranging from squamoid to mildly to moderately hyperplastic. The pathogenesis of goiter in the bongo may reflect a mixture of genetic predisposition coupled with environmental factors, including a period of exposure to a goitrogen.

MEMBRANE GLYCOLIPIDS AND THEIR RELATIONSHIP TO THE STRUCTURE AND FUNCTION OF CELL SURFACE RECEPTORS FOR GLYCOPROTEIN HORMONES, BACTERIAL TOXINS, AND INTERFERON

Gangliosides inhibit 125 I-thyrotropin (TSH) binding to the thyrotropin receptor. This inhibition, which appears to be hormonally specific, is critically altered by the number and location of the sialic acid residues within the ganglioside structure. The inhibition results from the interaction of gangliosides with TSH, rather than with the membrane receptor. The ganglioside-TSH interaction is associated with a distinct conformational change of the TSH molecule. 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 G M3 are present in bovine thyroid membranes in much higher quantities than have been previously found in extraneural tissue and are absent in a thyroid tumor with no TSH receptor activity. The finding that the B protein of cholera toxin, which also interacts with gangliosides, has a peptide sequence in common with the β sub-unit of TSH, suggests that TSH and cholera toxin may be analogous in their mode of action. It is suggested, therefore, that a ganglioside or ganglioside-like structure is a basic component of the thyrotopin receptor and that cholera toxin and TSH have a common mechanism by which their message is transmitted to the cell machinery.