Sulfonylurea binding to a low-affinity site inhibits the Na/K-ATPase and the KATP channel in insulin-secreting cells.

We have used hamster insulinoma tumor (HIT) cells, an insulin-secreting tumor cell line, to investigate modulation of the Na/K-ATPase and of the ATP-sensitive K channel (K(ATP)) by the sulfonylurea glyburide. Membrane proteins from cells cultured in RPMI with 11 mM glucose have at least two glyburide receptor populations, as evidenced by high and low binding affinity constants, (K(d) = 0.96 and 91 nM, respectively). In these cells K(ATP) channel activity was blocked by low glyburide concentrations, IC(50) = 5.4 nM. At 12.5 nM glyburide the inhibition developed slowly, tau = 380 s, and caused reduction of channel activity by 75 percent. At higher concentrations, however, inhibition occurred at a fast rate, tau = 42 s at 100 nM, and was almost complete. Na/K-ATPase activity measured enzymatically and electrophysiologically was also suppressed by glyburide, but higher concentrations were needed, IC(50) = 20-40 nM. Inhibition occurred rapidly, tau = 30 s at 50 nM, when maximum, activity was reduced by 40 percent. By contrast, cells cultured in RPMI supplemented with 25 mM glucose exhibit a single receptor population binding glyburide with low affinity, K(d)= 68 nM. In these cells inhibition of the Na/K-ATPase by the sulfonylurea was similar to that observed in cells cultured in 11 mM glucose, but K(ATP) channel inhibition was markedly altered. Inhibition occurred only at high concentrations of glyburide and at a fast rate; maximum inhibition was observed at 100 nM. Based on these data, we propose that glyburide binding to the high affinity site affects primarily K(ATP) channel activity, while interaction with the low affinity site inhibits both Na/K-ATPase and K(ATP) channel activities. The latter observation suggests possible functional interactions between the Na/K-ATPase and the K(ATP) channel.

Impairment of hypothalamic-pituitary-thyroid function in rats treated with human recombinant tumor necrosis factor-alpha (cachectin).

Tumor necrosis factor-alpha (TNF; cachectin), a peptide secreted from stimulated macrophages, mediates some of the metabolic derangements in inflammatory and neoplastic disorders. To determine whether TNF is responsible for the changes in hypothalamic-pituitary-thyroid (HPT) function in nonthyroid illnesses, we administered synthetic human TNF to male Sprague-Dawley rats. The rats were given TNF or saline (control; both pair fed and nonpair fed) iv (six to eight per group). HPT function was tested 8 h after administration of 200 micrograms TNF/kg BW, 8 h after 5 days of 150 micrograms TNF/kg BW, and 8 h after a 3-day series of 50, 200, and 800 micrograms TNF/kg BW. The single injection of 200 micrograms TNF/kg significantly reduced (all P less than 0.05) serum TSH, T4, free T4, T3, and hypothalamic TRH compared to the corresponding hormone levels in saline-injected control rats. Serum TSH and hypothalamic TRH recovered to normal levels after 5 days of 150 micrograms/kg TNF treatment. With the increasing daily doses of TNF, serum TSH and hypothalamic TRH fell significantly. Hepatic 5'-deiodinase activity was reduced after 1 day of TNF treatment, but increased after the 3-day series of injections. TNF treatment reduced pituitary TSH beta mRNA, but did not affect alpha-subunit mRNA. TNF treatment also reduced thyroid 125I uptake and reduced thyroidal release of T4 and T3 in response to bovine TSH, but did not change the TSH response to TRH. TNF treatment reduced the binding of pituitary TSH to Concanavalin-A, indicating that it alters the glycosylation of TSH. The TSH with reduced affinity for this lectin had reduced biological activity when tested in cultured FRTL-5 rat thyroid cells. In vitro, TNF inhibited 125I uptake by cultured FRTL-5 rat thyroid cells and blocked the stimulation of [3H]thymidine uptake by these cells. The data indicate that TNF acts on the HPT axis at multiple levels and suggest that TNF is one of the mediators responsible for alterations in thyroid function tests in patients with nonthyroidal illnesses.

Triac reduces serum TSH without decreasing alpha and beta TSH messenger RNAs.

Triac, 3,5,3'-triiodothyroacetic acid, was administered at doses of 1, 3, 9 or 30 micrograms/100 g body weight to hypothyroid rats to determine its effects on TSH secretion and pituitary mRNA content. Triac caused a dose-dependent decrease in serum TSH 6 h after injection. Pituitary content of mRNA subunits either remained at hypothyroid levels or increased at 6 h. At 24 h after injection of the 3 micrograms dose of triac, serum TSH returned to hypothyroid levels; both alpha and beta mRNA subunits were reduced at this time. When 30 micrograms triac/100 g body weight was administered, serum TSH levels remained depressed 24 h later, while pituitary mRNA content was essentially the same as in the hypothyroid controls. These findings indicate that the initial decrease in TSH secretion in response to triac is independent of the availability of TSH mRNA transcripts and suggest that TSH secretion and synthesis may be differentially controlled.

Hormonal control of thyrotropin and growth hormone secretion in a human thyrotrope pituitary adenoma studied in vitro.

Pituitary thyrotrope tumours are a rare cause of hyperthyroidism. Prior in vitro studies of these tumours have revealed various patterns of differentiation and secretory activity. We have characterized the histological, biochemical, molecular and physiological features of a thyrotrope adenoma in order to define its origin and autonomy. Histochemical and electron micrograph findings confirmed the diagnosis of a thyrotrope cell adenoma. Immunostaining was positive for TSH and GH in the cytoplasm of the adenoma cells. Tissue extracts contained TSH-IR which co-eluted with authentic hTSH when analysed by gel filtration. Tumour fragments studied in a tissue culture system secreted TSH, alpha-subunit and GH. TRH (30 nmol/l) stimulated TSH and GH secretion. T3 (1.5 nmol/l) inhibited GH release and had no effect on TSH secretion. GnRH (50 nmol/l), dexamethasone (10(-4) mol/l), SRIH (1 mumol/l) and TRH-glycine, a tetrapeptide precursor of TRH, stimulated TSH release. Dexamethasone inhibited GH and alpha-subunit secretion. Stable transcripts for alpha- and beta-subunits of TSH and GH messenger RNAs were detected by molecular hybridization in cytosolic fractions. Immunohistochemistry, in vitro secretory function, and mRNA analysis suggest multidirectional differentiation of the tumour cells. TRH-glycine may have a direct stimulatory effect upon pituitary thyrotropes.

Human chorionic gonadotropin stimulates iodide uptake, adenylate cyclase, and deoxyribonucleic acid synthesis in cultured rat thyroid cells.

hCG stimulates thyroid function, but it has been suggested that it is impurities in commercial hCG preparations or a variant of hCG that are responsible for the thyrotropic activity. In this study, we tested the thyrotropic activity of purified and commercial hCG and compared its action with that of bovine TSH (bTSH) in cultured rat FRTL-5 cells in regard to stimulation of iodide uptake, activation of adenylate cyclase, and synthesis of DNA. Iodide uptake was measured after incubation of the cells for 48-72 h with the test hormones, followed by a 40-min incubation with 0.1 microCi Na125I and 10 mumol/L carrier NaI; the 125I in the washed cells was counted. Adenylate cyclase was measured after incubation of the cells with the test stimulators for 3 h in hypotonic medium by RIA of cAMP in the medium. DNA synthesis was measured after incubation of the cells with the test substances for 24 h, followed by addition of [3H]thymidine for 3 h and then measuring the incorporation of [3H]thymidine into the cells. Both purified and commercial hCG produced a dose-related increase in iodide uptake. The relative potency of commercial hCG was 0.024 microU bTSH/U hCG and that of purified hCG was 0.042 microU bTSH/U hCG; compared with human TSH, the potency of purified hCG was 0.72 microU/U hCG. hCG caused a dose-related increment of adenylate cyclase and [3H]thymidine incorporation. The effect of hCG on iodide uptake and [3H]thymidine incorporation was additive with that of bTSH; hCG was not an antagonist of TSH in these cultured rat thyroid cells. We conclude that hCG has intrinsic thyrotropic activity in FRTL-5 cells in regard to stimulation of iodide uptake, activation of adenylate cyclase, and stimulation of DNA synthesis.

Triiodothyronine inhibits phorbol ester-induced thyrotropin release from elutriated bovine thyrotrophs.

The inhibitory effect of T3 on TSH release was studied on a population of thyrotroph-enriched cells prepared from bovine pituitary glands by centrifugal elutriation. The cells (2.0 X 10(5)/ml) were cultured for 2 days and then exposed to TRH, phorbol-12 myristate-13 acetate (PMA), or calcium ionophore (A23187) with or without 100 nM T3 for two different preincubation periods, 3 h and 24 h. Cytosolic TSH and release of TSH into the medium were measured by a specific RIA for bovine TSH. TRH (10 nM, 100 nM), PMA (100 nM, 1 microM, 10 microM), and A23187 (100 nM, 1 microM, 10 microM) increased TSH release in a dose-dependent manner. One-hundred nanomolar TRH, 10 microM PMA, and 10 microM A23187 increased TSH release maximally from 176 +/- 6 microU/ml (mean +/- SD, n = 4) to 240 +/- 40, 308 +/- 39, and 228 +/- 16, respectively. PMA and A23187 interacted synergistically in the release of TSH. Cytosolic TSH was not affected by TRH or A23187. PMA (100 nM) together with A23187 resulted in a decrease in cytosolic TSH. PMA alone (1 and 10 microM) decreased cytosolic TSH content to 84% and 77%, respectively, of the control level, suggesting that PMA enhances release of TSH. One-hundred nanomolar T3 had no effect on the basal release of TSH when given for 3 h, but resulted in a 47% decrease when administered for 24 h. The inhibitory effect of T3 on TRH-induced TSH release was found when the cells were preincubated with T3 for 24 h, but not for 3 h. In contrast, PMA-induced TSH release was significantly inhibited to 74% of induced levels by preincubation with T3 even for 3 h, and further inhibition occurred with an increase in preincubation time. These data suggest that the effectiveness of T3 depends on the mode of stimulation, and that the more immediate reaction observed with PMA induction may result from the interaction of T3 with the protein kinase C pathway.

Hypothalamic secretion of thyrotropin releasing hormone declines in aging rats.

Young and aged male rats were used in experiments to investigate a possible decline in hypothalamic secretion of thyrotropin releasing hormone (TRH) to the anterior pituitary of aging mammals. We observed a 66% decrease in basal TRH release by incubated rat hypothalami with aging. Thyroid hormone-responsive hepatic alpha-glycerophosphate dehydrogenase (GPD) and malic enzyme (ME) levels in aged rats did not differ from 5-month-old controls in spite of a significant fall in serum thyroxine (T4) levels with aging. Other results suggest that these particular indicators of thyroidal status should not change in the aging rat because serum T3 is maintained in the normal range. Serum thyrotropin (TSH) levels, which normally rise when serum T4 levels decline, did not change with aging. These data suggest that gradual loss of the essential TRH stimulation of TSH release with aging may be compensated for by a decline in T4 inhibition of TSH release at the pituitary.

Influence of thyroidal status on pituitary content of thyrotropin beta- and alpha-subunit, growth hormone, and prolactin messenger ribonucleic acids.

Thyroidectomized rats were used to study the effects of a single injection of T3 on pituitary mRNA synthesis and hormone secretion. T3 was injected ip at doses of 0, 0.2, 1, or 5 micrograms/100 g body weight, and and animals were killed 24 h later. T3 caused a significant decrease in serum TSH, but caused no significant change in either serum GH or PRL. Pituitary mRNA was quantified by slot blot hybridization with cDNA probes specific for alpha-TSH, beta-TSH, PRL, and GH. We found that both the alpha and beta mRNA subunits decreased, that PRL mRNA remained relatively unchanged, and that GH mRNA increased with increasing T3 dose. The data show that a single dose of T3 can profoundly influence mRNA levels in the anterior pituitary; the lowest dose of T3 caused maximum inhibition of alpha-TSH mRNA while beta-TSH mRNA declined further in a dose-dependent manner.

Differential expression of thyrotropin hormone genes by GH3 cells and the normal rat pituitary.

RNAs from GH3 cells, a rat clonal cell line, and anterior pituitaries of normal rats have been isolated and assayed for the presence of transcripts coding for the alpha- and beta-subunits of thyrotropin hormone (TSH) by hybridization to their respective cDNAs. Northern analysis indicated that GH3 cells lack both TSH transcripts, and that normal anterior pituitary cells contain mRNA for both the alpha- and beta-subunits approximating 800 and 700 nucleotides, respectively. An examination of the DNAs from GH3 cells and normal anterior pituitary tissue revealed no organizational difference when the restriction digests were subjected to Southern analysis. The lack of TSH secretion by GH3 cells is probably not due to sequence modification of genomic DNA, but to undetermined factors controlling transcription.

The effect of estrogen on the concentration of ovalbumin gene sequence in the 2 M NaCl residual fraction of oviduct chromatin.

Oviduct chromatin was isolated from both estrogenized and non-estrogenized hens. Extraction of the chromatin with 2 M NaCl removed a majority of the proteins, and the resulting DNA was then separated into two components: (1) a major fraction which was virtually protein-free; and (2) a minor fraction which was complexed with proteins. It was found that the DNA fraction that is complexed with proteins contained ovalbumin gene sequences and that the concentration of these sequences could be boosted by estrogen-treatment.

Simple isolation of DNA hydrophobically complexed with presumed gene regulatory proteins (M3).

Chromatin from chicken reticulocytes and mouse Ehrlich ascites tumor cells has been extracted with 2 M NaCl, leaving a portion of the DNA still complexed with a fraction of nonhistones (designated M3, since it can be dissociated from DNA in solutions of 3 M NaCl containing 5 M urea). The DNA complexed with M3, separated from the bulk DNA by centrifugation, was found to contain sequences poorly represented in bulk DNA. Specifically we found that DNA--M3 complexes isolated from chicken reticulocyte chromatin were enriched in globin gene sequences by 20-fold relative to unfractionated DNA and by over 1000-fold relative to DNA rendered free of protein following the extraction of chromatin with 2 M NaCl. We have therefore isolated DNA fractions complexed with M3 which are enriched in specific sequences as may be determined by M3.