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Interleukin-6 (IL-6), a pleiotropic cytokine, is postulated to be involved in the pathogenesis of sick euthyroid syndrome, although the direct in vitro effects of IL-6 on human thyroid function are controversial. Because IL-6 signal can be transduced when the complex of IL-6 and soluble IL-6 receptor (sIL-6R) binds to gp 130, an IL-6 signal transducer, we studied the effects of IL-6 and sIL-6R on thyroid function, using human thyroid follicles obtained from patients with Graves' disease. IL-6 alone had no inhibitory effect on TSH-induced thyroid function (125I incorporation and organic 125I release), even at supraphysiological concentrations. However, in the presence of physiological concentrations of sIL-6R (100 ng/ml), IL-6 inhibited thyroid function dose dependently and completely, accompanied with the decreased ratio of 125I-T3/125I-T4 not only in the thyroid follicles but also in the culture medium. Thyroid follicles did not secrete sIL-6R but produced IL-6 constitutively. Consistent with these findings, sIL-6R inhibited thyroid function slightly at high concentrations. Furthermore, RT-PCR analyses revealed that human thyroid follicles expressed the messenger RNAs for IL-6 and gp130 but scarcely messenger RNA for IL-6R. These in vitro findings suggest that IL-6 alone hardly affects thyroid function in thyroid follicles in which IL-6R gene is scarcely expressed. However, because sIL-6R is present abundantly in serum, IL-6 in vivo would be capable of inhibiting the synthesis and release of T4 and, to a greater extent, T3 from the thyroid gland. These in vitro findings are at least partly related to the development of sick euthyroid syndrome.

Basic fibroblast growth factor (bFGF) increases DNA synthesis in rat FRTL-5 thyroid cells, as measured by increased incorporation of tritiated thymidine into DNA. We show that this action is associated with the ability of bFGF to increase cytosolic Ca2+ levels and transiently increase c-fos mRNA levels. Other agents that increase c-fos mRNA levels and DNA synthesis in FRTL-5 cells include TSH, insulin, insulin-like growth factor-I, phorbol esters, A23187, and alpha 1-adrenergic agents; the last two agents also act by increasing cytosolic Ca2+ levels. Despite its enhancement of DNA synthesis, however, bFGF decreases TSH-induced cAMP-mediated iodide uptake. This action appears to reflect two separate actions of bFGF. First, bFGF decreases TSH receptor mRNA levels and the ability of TSH to acutely increase cAMP levels in FRTL-5 cells. The ability of bFGF to negatively regulate TSH receptor mRNA levels is additive to and independent of the ability of TSH and its cAMP signal to negatively autoregulate TSH receptor mRNA levels. This is consistent with the effect of bFGF on cytosolic Ca2+ levels and the ability of increased cytosolic Ca2+ to decrease TSH receptor mRNA levels. Second, bFGF inhibits cAMP signal expression, as evidenced by its ability to inhibit (Bu)2cAMP-induced iodide uptake in FRTL-5 cells. Both effects are, presumably, associated with the ability of bFGF to counteract TSH/cAMP-induced increases in thyroid peroxidase mRNA levels, which we demonstrate. We suggest, therefore, that bFGF causes opposite effects on DNA synthesis and iodide uptake because of its effect on cytosolic Ca2+ levels and because increases in cytosolic Ca2+ can have opposite effects on gene transcription, particularly in the case of the TSH receptor and c-fos genes.

Effects of retinoids on DNA synthesis, iodine metabolism, and thyroid peroxidase messenger RNA levels were studied in cultured porcine thyroid cells. Retinol (10(-8)-10(-5) M) alone did not affect DNA synthesis but potentiated that induced by epidermal growth factor or insulin-like growth factor-I without changes in the number or affinity of receptors for the growth factors, suggesting that retinol stimulates postreceptor events responsible for DNA synthesis. Retinol was an inhibitor of TSH-stimulated iodine metabolism. Iodide uptake and release of organified iodine stimulated by TSH or forskolin were inhibited dose dependently by treatment with retinol. The inhibition was detected at 10(-8) M and was approximately 50% at 10(-6) M. The potency of retinoic acid was comparable to that of retinol. The inhibitory effect of retinol was detected after treatments of thyroid cells for 24 h, and the maximal effect occurred after 48 h incubation. The cAMP accumulation in cultures treated with TSH plus retinol was lower than that of control cultures treated with TSH alone. However, iodide uptake stimulated by 8-bromo-cAMP was also inhibited by retinoids. Retinol reduced TSH- or 8-bromo-cAMP-stimulated gene expression of thyroid peroxidase. Thus, the data suggest that retinoids inhibit TSH-stimulated iodine metabolism by reducing cAMP accumulation and also by acting on the steps subsequent to cAMP production.

Methimazole (MMI) increases thyroglobulin (Tg) mRNA levels in FRTL-5 rat thyroid cells. The increase reflects a transcriptional action of the antithyroid agent and is inhibited by cycloheximide, as is the transcriptional action of TSH. It takes several hours to be apparent, is maximal between 24-48 h, and is specific, in that thyroid peroxidase and beta-actin mRNA levels are not increased simultaneously. The increased mRNA levels are associated with increased recovery of immunoprecipitable Tg in the medium of cells exposed to [35S]methionine. The MMI effect appears to be independent of the action of TSH or its cAMP signal, since the MMI-induced increase in Tg mRNA levels is evident in cells treated with TSH or maintained in its absence and is associated not with increases in cAMP levels but, rather, under some circumstances with a decrease. The effect is evident under conditions in which the ability of insulin or insulin-like growth factor-I to increase Tg mRNA levels is already maximal. The MMI-induced increase is inhibited by concentrations of iodide associated with autoregulation of FRTL-5 rat thyroid cells, is inhibited but not mimicked by propylthiouracil, and is not altered by T3. The increase in Tg mRNA levels does not correlate with increased DNA synthesis as a function of MMI concentration either in cells treated with TSH or in those maintained in its absence. A concentration of MMI (5 mM) that increases Tg mRNA levels can also inhibit 8-bromo-cAMP- or phorbol ester-induced increases in [3H]thymidine incorporation into DNA.

A 73-yr-old patient with idiopathic hypoparathyroidism was admitted to our hospital in May 1981. The immunoreactive PTH (iPTH) level determined by RIA using antiserum specific for the C-terminal region of PTH-(65-69) was in the upper normal range (0.6 ng/mL) and over the next 7 yr increased gradually to 6 ng/mL. Since iPTH levels determined using other commercial RIA kits remained constantly decreased or in the undetectable range, we studied the mechanism of false elevation of iPTH in this patient. The patient's serum contained no binding protein to the tracer ([125I]) [Tyr45] human PTH-(46-84)), nor was any heterophilic antibody to the first [guinea pig immunoglobulin G (IgG)] or the second antibody (goat IgG) detected. Consistent with these findings, the dilution curve of the serum was parallel with that of standard bovine PTH-(1-84). Gel filtration analysis revealed that the iPTH-like substance was eluted in the void volume (apparent mol wt, greater than 70,000). Almost all of the iPTH-like substance was adsorbod by a protein-A-Sepharose column. When the IgG fraction purified by protein-A-Sepharose affinity chromatography was applied to an antihuman IgG lambda-Sepharose column, 72% of the iPTH-like substance was detected in the IgG lambda. These results suggest that the falsely elevated iPTH in the patient's serum was due to IgGs (mainly IgG lambda), which were cross-reactive with the antiserum highly specific for the C-terminal region of human PTH-(65-69).

The serum levels of somatomedin A, as measured by radioreceptor assay, were significantly reduced in rats 2 days after the administration of streptozotocin. The mean decrease was 45.4 +/- 2.9% of the initial values. In rats treated with insulin, blood glucose levels and glycosuria decreased, and serum somatomedin A returned to 108.3% +/- 11.7% of the initial values by the sixth day of treatment. In untreated diabetic rats, serum somatomedin A decreased progressively to 23.4 +/- 4.4% 8 days after streptozotocin administration. The total caloric intakes in the treated and nontreated diabetic rats were similar, suggesting that the low levels of somatomedin A in diabetic rats may be due to lack of insulin. A significant correlation was observed between serum somatomedin A values and body weight (r = 0.90) or the urinary glucose (r = -0.84) or blood glucose levels (r = -0.67). When the diabetic insulin-treated rats were fed a low protein diet, there was no increase in serum somatomedin A. Inhibitory factors in serum which interfere in the bioassay for somatomedin had no effect in our radioreceptor assay.

Direct effects of cyproheptadine, reserpine, synthetic ovine corticotropin-releasing factor (CRF), dexamethasone, and lysine-8-vasopressin (LVP) on the secretion of immunoreactive ACTH and beta-endorphin from the adenoma and the nonadenomatous tissue of patients with Cushing's disease were examined using a superfusion system. Cyproheptadine and reserpine (10(-9)-10(-7) M of each) suppressed immunoreactive ACTH and beta-endorphin secretion from both tissues. CRF (10(10)-10(7) M) stimulated the secretion of both peptides from the nonadenomatous tissue, but only a high dose of CRF could stimulate the secretion of these peptides from some adenomas. Such CRF-induced secretion was partially suppressed by dexamethasone. LVP (10(-9)-10(-7) M) stimulated peptide secretion from both types of tissue. These results suggest direct inhibitory effects of cyproheptadine and reserpine on the secretion of these peptides from the pituitary of patients with Cushing's disease, a different stimulatory mechanism of LVP from that of CRF in these tissues, and low sensitivity of the adenoma to CRF.

[125I]Insulin-like growth factor I (IGF-I) specifically bound to erythrocytes; the binding was saturable, and time and temperature dependent. Steady state binding was reached at 16 h at 4 C, and specific binding averaged 14.3 +/- 0.7% (+/- SEM) at a concentration of 3.6 X 10(9) cells/ml in seven normal subjects. [125I]IGF-I binding to the cells was displaced by unlabeled IGF-I in a dose-dependent manner. Scatchard analysis indicated a linear plot, and Ka and number of binding sites/cell were 1.43 +/- 0.07 X 10(9) M-1 and 20.7 +/- 2.2, respectively. Compared to IGF-I, the relative potencies of multiplication-stimulating activity and insulin for displacing [125I]IGF-I binding were 20% and 1%, respectively. [125I]IGF-I binding to erythrocytes from patients with acromegaly was lower than binding to cells from pituitary dwarfs. An inverse correlation between plasma IGF-I level and the number of IGF-I-binding sites per cell was found (r = -0.75; P less than 0.005). These results demonstrate that [125I]IGF-I binding to erythrocytes can be used for clinical measurement of the IGF-I receptor.

To study the effect of acute stress on ACTH secretion and synthesis in rat pituitary and hypothalamus, ACTH content and POMC mRNA levels (measured by use of Northern blot analysis) in these tissues as well as the levels of ACTH in plasma and those of CRF in the hypothalamus were determined after insulin-induced hypoglycemia. Plasma ACTH levels increased at 30 and 60 min. ACTH levels in the anterior pituitary lobe (AP) decreased at 30 min, and then returned to control levels at 60 min. No change was seen in the intermediate-posterior pituitary (IP) or the hypothalamus after insulin injection. CRF levels decreased at 30 and 60 min, then returned to control levels at 90 min in the medial basal hypothalamus, including the median eminence. Hybridization with a cDNA probe revealed a single size class of POMC mRNA in AP, IP, and hypothalamus, and the size of POMC mRNA in these tissues did not change during the experimental period. POMC mRNA levels in AP increased at 60 min and reached a peak at 120 min, but those in IP and hypothalamus did not change. These results suggest that 1) insulin-induced hypoglycemia stimulates both secretion and synthesis of ACTH (at least by increasing POMC mRNA levels) in the AP, and 2) the levels of ACTH and POMC mRNA in the IP and hypothalamus are not affected by insulin-induced hypoglycemia.

The fate of cell-bound [125I]iodo-human GH ([125I]iodo-hGH) was studied in monolayer cultures of hepatocytes from pregnant and nonpregnant rats. Both the binding of [125I]iodo-hGH and its degradation were significantly higher in cells from pregnant than from nonpregnant rats. The positive correlation between the number of binding sites for hGH and the amount of degradation of [125I]iodo-hGH suggests that degradation, at least in part, is a receptor-mediated process. Degradation as a time- and temperature-dependent process was impaired by lysosomotropic compounds such as chloroquine and NH4Cl in a dose-dependent manner. Dinitrophenol, N-ethyl-maleimide, and NaN3 were also effective in preventing degradation, suggesting that an energy-requiring process is involved in degradation of [125I]iodo-hGH. Cell-bound [125I]iodo-hGH became less dissociable as a function of time of association. Degradation of [125I]iodo-hGH accelerated the dissociation of cell-bound radioactivity. Gel-filtration experiments revealed that [125I]iodo-hGH is degraded to smaller molecular species, but only a small portion of the degradation products exists within the cells. These observations suggest that receptor-bound [125I]iodo-hGH is degraded by an internalization process; lysosomal enzymes are probably responsible for the degradation of [125I]iodo-hGH, and the degraded products are rapidly released into the extracellular space.

Various aspects of thyroid hormone economy were examined in 15 patients with molar pregnancy and in 5 patients with choriocarcinoma. None of the patients with molar pregnancy was clinically thyrotoxic, though serum thyroxine (T4) was increased in 13 and free T4 concentrations were above normal in four of ten in whom measurements were made. In 2 patients with elevated serum T4 levels and in one patient with normal serum T4 levels, the daily rate of T4 disposal was increase, and most of 131I derived from 131I-T4 was excreted into urine in two patients in whom estimation of urinary excretion was made. Serum total triiodothyronine (T3) concentrations closely paralleled those of serum T4, but T3/T4 ratios were lower than those previously found in patients with toxic diffuse goiter of Graves' disease. Unresponsiveness to exogenous thyrotropin-releasing hormone (TRH) was observed in 5 patients with molar pregnancy whose serum total T4 and T3 levels were increased. Human chorionic gonadotropin (hCG) concentrations in serum ranged from 0.5 to 2830 IU/ml and were less than 200 IU/ml in 36% of the patients, possibly because molar pregnancy was diagnosed by ultrasonography, rather than high titers of urinary or serum hCG. Serum thyroid stimulating activity measured by the McKenzie bioassay closely paralleled values for hCG (r:+0.94). All thyroid abnormalities disappeared quickly after removal of the mole. In choriocarcinoma, no abnormality of thyroid function was found. These results suggest that: 1) some patients with molar pregnancy display a marked elevation of serum T4, T3, and free T4 concentrations, probably as a result of the action of a thyroid stimulator which is closely related to serum hCG or may be hCG itself; 2) T4 production rates are increased in these patients, and the pituitary responds to TRH in a manner similar to that found in thyrotoxicosis. Despite this, frank clinical thyrotoxicosis is usually absent, possibly because of relatively low serum T3/T4 ratios, possibly because of the limited duration of thyroid hyperfunction, and possibly because of other factors that for the present remain unknown.

Daily (24-h) urinary GH excretion was measured using a highly sensitive sandwich enzyme immunoassay in 10 normal adults, 6 patients with hypopituitarism, 25 normal but short children who had normal plasma GH responses (peak plasma GH level, greater than 10 micrograms/L) to provocative tests, and 8 patients with acromegaly. The mean urinary GH values in the normal adults, patients with acromegaly, and patients with hypopituitarism were 13.8 +/- 4.0 (+/- SE) and 431.1 +/- 149.1 ng/g creatinine (Cr) (1.56 +/- 0.45 and 48.77 +/- 16.87 ng/mmol Cr) and undetectable, respectively; these mean values were significantly different from each other. In the normal but short children the urinary values ranged from undetectable to 55.8 ng/g Cr (6.31 ng/mmol Cr). All of the normal but short children and 4 patients with hypopituitarism participated in a 24-h endogenous GH secretion study. The urinary GH values correlated significantly with the mean 24-h plasma GH concentrations as an index of endogenous GH secretion (r = 0.81; P less than 0.001) and plasma somatomedin-C levels (r = 0.67; P less than 0.001), respectively. In 6 patients with acromegaly whose plasma GH levels were constant throughout a 4-h period, the urinary GH values also significantly correlated with the mean plasma GH levels (r = 0.95; P less than 0.01). These data indicate that urinary GH measurements reflect endogenous GH secretion and that measurement of urinary GH excretion is a useful, simple, and practical method for evaluating endogenous GH secretion.

Kidney slices were obtained from castrated-hypophysectomized male rats treated with a single injection of several different gonadotropin preparations (two ovine LH fractions, one bovine LH fraction, and one hCG fraction) or vehicle, then incubated in a buffer containing [3H]thymidine. Only one of the above, an ovine LH preparation, increased [3H]thymidine incorporation into renal DNA, with a peak occurring 8-10 h after injection and therefore termed renotropin. However, in hypophysectomized rats with intact testes, such an effect was not observed. Furthermore, while testosterone propionate alone did not alter basal incorporation in castrated-hypophysectomized rats, it abolished the incorporation that was stimulated by renotropin. These results suggest that androgens, whether of testicular origin or exogenously administered, suppress the increased incorporation of [3H]thymidine stimulated by renotropin. This antagonistic effect of androgen was also observed with T4, but to a lesser degree. Our findings confirm the presence of renotropin, which could not be attributed to other known pituitary hormones, and suggest that there is a complex interaction between this factor and two other renal growth-promoting hormones, testosterone and T4.

D-Ala, Mephe, Met, enkephalin (Sandoz FK 33-824) is a stable long acting analog of methionine-enkephalin. FK 33-824 (0.5 or 1.0 mg), elicited plasma GH and PRL responses in normal subjects. In 23 patients with pituitary dwarfism, the response of plasma GH was markedly impaired, while PRL responded to a variable degree. In patients with acromegaly, there was little or no increase in GH and PRL after FK 33-824. Plasma GH increased to a variable degree after FK 33-824 in patients with hyperprolactinemia, with little change in plasma PRL. FK 33-824 decreased plasma cortisol in normal subjects and patients with pituitary disease. These results show that patients with acromegaly and hyperprolactinemia due to pituitary adenomas and patients with pituitary dwarfism do not respond well to FK 33-824, presumably because of hypothalamic or pituitary derangement.

We have examined the effect of glucose and FFA on GH-releasing factor (GHRF)-mediated GH secretion in rats under pentobarbital anesthesia. Hyperglycemia did not affect GH secretion induced by administration of 20, 100, and 200 ng GHRF/100 g body weight. In contrast, GH response to 50 ng GHRF/100 g body weight in lipid heparin-treated rats, which showed high plasma FFA levels, was significantly suppressed compared with the control group (plasma peak GH: control, 1526 +/- 263 ng/ml; lipid-heparin group, 377 +/- 69 ng/ml P less than 0.05, mean +/- SEM). This suppressive effect of FFA on GH secretion was abolished by pretreatment with antisomatostatin serum (ASS) (GH level at 4 min after GHRF administration: ASS-saline group, 1606 +/- 210 ng/ml; ASS-lipid-heparin group, 1531 +/- 174 ng/ml; mean +/- SEM). These results suggest that hyperglycemia does not change the GH response to GHRF and that elevation of plasma FFA suppresses GHRF-induced GH secretion by the stimulation of somatostatin secretion in rats.

We studied gonadotropin hormone alpha-subunit and gonadotropin secretion in four patients with gonadotropin-producing pituitary adenomas. All four patients had elevated plasma alpha-subunit levels, ranging from 2.8-8.5 ng/ml (normal, less than 0.5 ng/ml). alpha-Subunit responses to LHRH were less than those in seven patients with primary gonadal failure. The relative proportions of the gonadotropin and alpha-subunit peaks in one patient were the same before and after LHRH administration, based on gel filtration studies of plasma. The alpha-subunit levels decreased little during testosterone treatment in the two adenoma patients so treated. Immunohistochemical study of the adenomas from two patients demonstrated definite staining with alpha-subunit and gonadotropin antisera. Elevated plasma levels of alpha-subunit and its relative unresponsiveness to LHRH stimulation or testosterone suppression suggest that the alpha-subunit originated in tumor tissue and that its measurement is useful for the diagnosis of a gonadotropin-producing tumor in patients with elevated plasma levels of LH and/or FSH.

In the rat pituitary gland, immunoreactive angiotensin II (ANG II), renin, and LH, but not PRL, were found within the same cells of the anterior pituitary gland by staining with the avidin-biotin complex method in adjacent sections. No renin-positive staining was observed in the pituitary of the rats after 10 days of castration, but positive staining reappeared after 8 weeks. This effect of castration on renin immunoreactivity was abolished by the simultaneous administration of testosterone. In contrast, ANG II immunoreactivity was unaffected by castration. The intensity of renin immunoreactivity in the pituitary was less prominent in the female than in the male rat. These results suggest that there exists a pituitary renin-angiotensin system localized in the gonadotrophs and that the pituitary renin is under androgenic control.

A specific RIA for human pancreatic tumor GH-releasing hormone [hpGRH(1-44)NH2] was developed using an antiserum which recognizes the region (Met27-Leu44)NH2 of hpGRH(1-44)NH2. This RIA was used to measure GH-releasing hormone-like immunoreactivity (GRH-LI) in various human tissue extracts. The highest concentration of GRH-LI was detected in extract of pituitary stalk with moderate amounts found in hypothalamus and optic chiasm but none in the cerebrum, cerebellum, medulla oblongata, spinal cord, and anterior pituitary. In peripheral organs appreciable quantities of GRH-LI were found in the pancreas, whereas extracts of thyroid, lung, stomach, duodenum, ileum, colon, adrenal, and kidney contained very low concentrations of GRH-LI. No GRH-LI was detected in liver and spleen extracts. Gel permeation chromatographic analysis of the tissue extract from the hypothalamus revealed only one peak which eluted at the same position as that of 125I-hpGRH(1-44)NH2. Similar analysis of an extract from the optic chiasm showed one peak at the same position as that of 125I-hpGRH(1-44)NH2 and another peak which eluted after hpGRH(1-44)NH2. In contrast, an extract from the pancreas contained only one peak which eluted before 125I-hpGRH(1-44)NH2, indicating a possible precursor form of hpGRH(1-44)NH2. Limited trypsin digestion of the GRH-LI material from the pancreas, followed by gel permeation chromatographic analysis, yielded a major peak eluting at the same position as that of 125I-hpGRH(1-44)NH2. These results suggest that the GRH-LI detected in the hypothalamus most likely corresponds to hpGRH(1-44)NH2 in structure and that the GRH biosynthesized in the hypothalamus is transported to the stalk median eminence and stored there for release into the portal vessels.

Corticotropin-releasing factor (CRF)-like activity in two different kinds of ACTH-producing tumors (human ectopic ACTH-producing colonic cancer and rat MtT/F4 tumor) was determined by an in vitro method using isolated normal rat pituitary cells. The ACTH content of the post mortem colonic cancer was 5.5 ng/g w.wt. The ACTH content of the medium of MtT/F4 tumor cells was 153+/-32 pg/10(5) cells. The ACTH content of MtT/F4 tumor cell suspensions was elevated with increasing doses of hypothalamic median eminence extract (HME). The response of MtT/F4 tumor cells to HME was suppressed by 1 mug/ml of dexamethasone. Extracts of colonic cancer, MtT/F4 tumor and HME produced elevation of the ACTH content of the medium of isolated rat pituitary cells. The CRF-like activities of two kinds of tumor extracts in multiple dilutions ran parallel to that of HME. The CRF-like activities were 0.037 HME equiv/mg w.wt in MtT/F4 tumor and 0.052 HME equiv/mg w.wt. in colonic cancer. These results demonstrated that CRF-like activity existed in these two kinds of ACTH-producing tumors.

To study the effect of acute stress on CRF release and synthesis in rat hypothalamus, ACTH levels in plasma, CRF contents in the median eminence (ME), and CRF mRNA levels in the hypothalamus without ME and cerebral cortex were determined after insulin-induced hypoglycemia. Plasma ACTH levels increased at 30 and 60 min, while ME CRF content decreased at 30 and 60 min, then returned to the control level at 90 min. Hybridization with a cRNA probe revealed a single size class of CRF mRNA in the hypothalamus and cerebral cortex (approximately 1300 nucleotides), and the size of CRF mRNA in these tissues did not change during the experimental period. CRF mRNA levels in the hypothalamus increased to 130% of the control value at 30 min and reached a peak (186% of the control value) at 120 min, but these levels in the cerebral cortex did not change. These results suggest that insulin-induced hypoglycemia stimulates CRF synthesis by increasing CRF mRNA levels in the hypothalamus as well as CRF release, and that release and synthesis of CRF in the cerebral cortex are independent of those in the hypothalamus.

The effect of transforming growth factor (TGF)-beta on DNA synthesis and iodine metabolism was studied in cultured porcine thyroid cells. TGF-beta dose-dependently inhibited DNA synthesis stimulated by both insulin-like growth factor I and epidermal growth factor but did not affect the number or affinity of receptors for the two growth factors, suggesting that TGF-beta inhibits postreceptor events responsible for initiation of DNA synthesis. TGF-beta was a potent inhibitor of iodine metabolism. When porcine thyroid cells were cultured with TSH for 3 days in the presence of TGF-beta, TSH-induced iodide uptake and organification were reduced at rates that were dependent on the TGF-beta concentrations. The inhibition was detectable at TGF-beta concentrations as low as 50 pg/ml, and complete suppression was seen at 1 ng/ml. Only 6 h of exposure to TGF-beta resulted in a significant inhibition of TSH-induced iodine metabolism. Treatment of thyroid cells with TGF-beta for 3 days did not reduce cAMP production stimulated by TSH. Moreover, the intracellular cAMP level of thyroid cells cultured with TSH plus TGF-beta did not differ from that of cells cultured with TSH alone. TGF-beta decreased iodide uptake stimulated by forskolin or 8-bromo-cAMP. These results strongly suggest that TGF-beta inhibits TSH-stimulated iodine metabolism, at least in part, by affecting events subsequent to cAMP production. The physiological role of TGF-beta remains to be determined, but it may be involved in the regulation of thyroid cell growth and function.

One hundred and sixty-one children with idiopathic GH deficiency who received GH treatment were followed until they reached their final height. Final height was found to be influenced by gonadal function. In 108 patients who had spontaneous puberty (91 boys and 17 girls; group A), the mean final height was 151.8 +/- 6.6 (+/- SD) cm in boys and 141.7 +/- 7.4 cm in girls. In 29 patients with combined GH and gonadotropin deficiency (23 boys and 6 girls; group C), whose pubertal development was induced artificially at age 19.5 +/- 2.1 yr in the boys and 18.6 +/- 1.8 yr in the girls, the mean final height was 163.7 +/- 3.9 cm in boys and 151.0 +/- 5.1 cm in girls. The differences in final height between groups A and C were significant in both boys and girls. The shorter final height in group A was caused by the shorter pubertal duration and smaller pubertal height gain than those in normal children. In 24 patients (17 boys and 7 girls; group B) who developed early signs of puberty, gonadal suppression therapy with cyproterone acetate and/or medroxyprogesterone acetate was given. The mean SD score of the final height in these 24 patients was -2.1 +/- 0.6, significantly higher than that in group A. This beneficial effect of gonadal suppression treatment on final height was caused by increases in the duration of puberty and the pubertal height gain.

Synthetic human pancreatic GRF (hpGRF-44) was administered as an iv bolus to 28 normal children with short stature and 27 patients with GH deficiency. After a dose of 1 or 2 micrograms hpGRF-44/kg BW, mean plasma GH levels peaked at 15 and 30 min, respectively, with corresponding values of 30.1 +/- 4.7 and 33.2 +/- 3.7 ( +/- SE) ng/ml in normal but short children. The overall plasma GH response was greater than that of other GH stimulation tests such as insulin-induced hypoglycemia, glucagon-propranolol or L-dopa administration. Plasma LH, FSH, TSH, PRL, and cortisol levels were not altered by hpGRF-44 injection. Sixteen of 27 patients with GH deficiency did not respond to a 2 micrograms/kg BW hpGRF-44. However, plasma GH increases to greater than 5 ng/ml occurred in the remaining 11 patients. Their GH levels reached peaks between 15 and 90 min, with values ranging between 5.8 and 17.8 ng/ml. Two of these responding patients were infused iv with hpGRF-44 at 2.5 micrograms/min for 90 min after receiving an iv bolus injection of 2 micrograms/kg BW. Their plasma GH levels increased and remained near peak values throughout the infusion period. However, no increase in plasma GH levels occurred after a second bolus injection of hpGRF-44 given at the end of the infusion. These results suggest that hpGRF-44 is useful for the diagnosis of GH deficiency in individuals with short stature and that some patients with GH deficiency, diagnosed on the basis of established tests, have GH responses to hpGRF-44.

To elucidate the mechanism of humoral hypercalcemia elicited by human esophageal carcinoma cells (EC-GI), which constitutively produced interleukin-1 alpha (IL-1 alpha) and PTH-like factor, the effects of IL-1 alpha and PTH-related protein (PTH-rP) on bone resorption in vitro and on serum calcium concentrations in vivo were investigated. Nude mice transplanted with EC-GI cells invariably developed hypercalcemia, although their urinary cAMP excretion remained within the normal range. IL-1 alpha or PTH-rP-(1-34) stimulated 45Ca release from prelabeled fetal mouse forearm bones in a concentration-dependent manner, and when combined, IL-1 alpha and PTH-rP-(1-34) synergistically stimulated bone resorption in vitro. Injection of PTH-rP-(1-34) into mice three times a day for 2 days increased the serum calcium concentration in a dose-dependent manner. Continuous infusion of IL-1 alpha occasionally increased the serum calcium concentration. Simultaneous administration of IL-1 alpha at rates of 1-2.7 micrograms/day and PTH-rP-(1-34) at doses of 15-30 micrograms/day synergistically increased the serum calcium concentration in vivo. These findings suggest that PTH-rP and IL-1 alpha produced by the tumor cells were synergistically responsible for the humoral hypercalcemia observed in both the original patient and the tumor-bearing nude mice, and that at least two bone-resorbing factors [PTH-rP and another nonadenylate cyclase-stimulating bone-resorbing factor(s)] are active in patients with malignancy-associated hypercalcemia, in whom nephrogenous cAMP excretion is neither increased nor decreased.

The plasma GH response to GH-releasing hormone (GHRH), TRH, or GnRH administration was examined in 25 acromegalic patients. Plasma GH levels increased in 21 patients after GHRH, in 19 after TRH, and in 4 after GnRH. The four GHRH nonresponders had had acromegaly longer than had the GHRH responders. No specific combination of GH responsiveness to these 3 releasing hormones was found among the patients. Infusion of 1 mg GHRH for 150 min gradually increased plasma GH levels, with some fluctuations, from the beginning to the end of infusion in normal subjects and in 7 patients who were GHRH responders, but a bolus injection of 100 micrograms GHRH at the end of the infusion did not further elevate plasma GH levels. These results suggest that desensitization to GHRH occurred in the normal subjects and acromegalic patients. However, in 5 acromegalic patients who responded to both GHRH and TRH, a bolus injection of 500 micrograms TRH given at the end of the 150-min infusion of 1 mg GHRH evoked a further plasma GH rise. In 5 normal subjects and 2 patients who were responders to GHRH but not TRH, a bolus injection of 500 micrograms TRH did not cause plasma GH elevation at the end of 150-min infusion of 1 mg GHRH. These results imply that TRH and GnRH stimulate GH secretion from the adenoma cells in vivo through receptors different from those for GHRH. In vitro studies using cultured pituitary adenoma cells from 2 patients revealed that the responses of GH secretion to GHRH were similar to those in vivo. These data, therefore, suggest that the responsiveness of GH secretion to stimuli is determined by the specificity of the receptors on adenoma cells. The action of somatostatin-28 was more potent than that of somatostatin-14 in the suppression of GH secretion from adenoma cells.

The effect of plasmapheresis on thyroid hormone secretion was studied in the dog. Approximately 10% of body weight volume of blood was exchanged with red blood cells suspended in dextran solution over a period of 10-30 min. A striking increase in thyroid hormone release was observed within 2 hr. Addition of thyroid hormone to the blood cell suspension, however, did not prevent this response. Although the increase in thyroid hormone release was not universally seen in hypophysectomized dogs, in decerebrate dogs and in some hypophysectomized dogs a moderate response was observed. These results suggested the possibility that some mechanism other than pituitary-thyroid feedback might be involved. Since no marked change was observed local perfusion of the thyroid with blood cell suspension, extrathyroidal humoral factors were probably involved in this response. (Endocrinology 86: 1281, 1970)

IN RECENT years the relationship between hormones and the mechanism of pigmentation has come to the fore. In this report it will be shown that the pituitary gland elaborates a melanocyte-stimulating hormone (MSH) which affects melanin pigmentation in man. The “peculiar change” in skin color described by Addison in patients with adrenocortical insufficiency, the decreased pigmentation of panhypopituitarism, and the melasma of pregnancy seem to depend in large part upon the activity of MSH. Because the developments leading to this conclusion have involved many people and many aspects of research, it is worthwhile to review the historical background of this problem. In 1916 Smith and Allen, two biologists working independently, almost simultaneously reported that removal of the pituitary gland of tadpoles was followed by loss of skin color (1–4). Three years later Atwell showed that when tadpoles were immersed in pituitary extracts their skin became darker (5). Atwell, Smith, Swingle, and later Zondek found ...

The effect of an acute reduction of thyroid hormone level in the circulating blood on thyroid function has been studied in the dog. PBI and free thyroxine index in the blood of a recipient dog were decreased to approximately 70% of their previous levels by means of exchange transfusion from a thyroidectomized-hypophysectomized dog. Thyroid hormone secretion was estimated by determination of PB l31I concentration in thyroid venous blood. No increase in thyroid hormone secretion was observed within 4 hr in either chloralose anesthetized dog or curare immobilized dog. A long-term observation revealed that peripheral blood PBI recovered significantly 2 weeks after exchange transfusion, and free thyroxine index 5 days after. These results suggest that the feedback mechanism does not respond to a moderate reduction of circulating thyroid hormone in the dog immediately, but rather sluggishly over a period of days. (Endocrinology 84: 1161, 1969)

Growth hormone content in the pituitary gland was measured by radioimmunoassay in 48 human fetuses at various gestational stages. None of the fetuses of 6 weeks gestation contained detectable amounts of growth hormone. However, growth hormone was measurable in some fetuses starting at the 7th week, and by the 9th week it was detected in all fetuses studied. Thereafter, growth hormone content increased in excess of the body weight gain. Growth hormone concentration in cord blood was also studied in 7 fetuses. It ranged from 18 ng/ml in a 10-week-old to 120 ng/ml in a 4-month-old fetus. It was concluded that human fetuses synthesize and store growth hormone as early as the 7th gestational week.

ALTHOUGH periodic paralysis is a rare disorder, its association with hyperthyroidism has been reported by several investigators (1–15). In reviewing the literature, it was found that only about 30 such cases have been reported from the United States and Europe, whereas more than 70 cases have been reported from Japan. Possibly the association of these two disorders is less rare in Japan. As the Japanese reports were all concerned with relatively small numbers of patients with hyperthyroidism, the incidence of the association of these two disorders in Japan could not be estimated. In order to obtain a fairly good estimate, the incidence of periodic paralysis among 6,333 cases of hyperthyroidism operated upon in three thyroid hospitals in Japan was studied. These hospitals were located in different parts of country—Noguchi Hospital in Beppu, Kyushu (southern part of Japan), Kuma Hospital in Kobe (central part of Japan) and Ito Hospital in Tokyo (eastern part of Japan), so the study provided data on a cross ...

In order to observe the alterationsin intrathyroidal iodine metabolism which occur in response to chronic iodide administration, iodine metabolism in rats given 500 µg of iodide/ day (iodide group) was compared to that in rats given 10 μg of iodide/day (control group). Ratswere given 126I with 127I in their drinking water for 4 weeks until the time of sacrifice. Rats were also injected with 131I and killed at various time intervals up to 48 hr after 131I. In the 2 groups, thyroidal 127I content and serum iodothyronine- 127I concentrations did not differ significantly from each other, while absolute iodine uptake calculated from thyroidal clearance of 131I multiplied by the serum 127I concentration was 2.5 times greater in the iodide group than in the control. With respect to both m I and 131I, the. proportion of iodothyronines as a per cent of total thyroidal organic iodine did not differ greatly in the 2 groups. This indicated the apparently contradictory results that the iodide group took up 2.5 times a...

SINCE 1926, sporadic reports have appeared on the presence of the melanocyte-stimulating hormone (MSH) in mammalian urine and blood. Because many of these studies were performed with inadequate techniques, and because present-day methods make possible the quantitative detection of MSH in minute amounts with an in vitro bioassay method, it was decided to clarify this problem by determining the MSH in the urine and blood of patients with various disorders. REVIEW OF THE LITERATURE Urinary MSH. Collin and Drouet (1) observed the color change of intact frogs after the injection of human urine. Darkening was produced with urine from patients with pituitary tumors, hyperthyroidism, migraine, and retinal hemorrhage. Positive results also were obtained with the urine of normal females, which was obtained the day prior to, or on the first day of menstruation. Konsuloff (2) used hypophysectomized frogs and found that urine from 9 pregnant women contained large amounts of MSH. He suggested that the demonstration of ...

Experiments were performed to determine the effects of graded chronic doses of iodide on thyroid hormone synthesis. Rats were given a low iodine diet for a week and were then given a diet supplemented with graded doses of iodide (5-3645 μg of iodide in 20 g of diet) for 10 days until the time of sacrifice. Rats were also given a single intravenous injection of 131I 1 hr before sacrifice. 127I content of thyroid and plasma was determined by the method of Baker. Thyroidal total 127I uptake per unit of time was measured as thyroidal total 131I divided by the mean of the concentrations of inorganic 131I in plasma collected 2 min and 1 hr after the injection of 131I and multiplied by the concentration of inorganic m I in plasma collected 1 hr after 131I. Thyroidal organic 131I formed per unit of time was calculated from thyroidal organic 131I instead of thyroidal total 131I. These calculations could be expressed as thyroidal 131I clearance multiplied by plasma inorganic 127I or thyroidal 131I multiplied by the...

EEG, pulse rate, respiration and eye movement were polygraphically recorded throughout the night in 10 normal adult male subjects. Blood was collected every 20 min throughout the night by means of an indwelling venous catheter without disturbing the natural course of sleep. Human growth hormone (HGH) and blood sugar were measured in these plasma samples. Depth of sleep was classified into 5 stages by EEG. A marked elevation of plasma HGH was constantly observed coincident with the onset of sleep. Following the initial large HGH peak, additional peaks of HGH, with a decrease in the size of subsequent peaks, were observed. Shifting the time of the onset of sleep by 3 hr before or after the usual bedtime did not change the elevation of plasma HGH coincident with the onset of sleep. When a state of full wakefulness was maintained at night, plasma HGH did not rise. The secretion of plasma HGH was inhibited during paradoxical sleep. It is concluded that nocturnal sleep is a potent stimulator for the se...

By means of the technique of cannulation of the thyroid veins of dogs, the present experiments were undertaken to investigate the nature of nonhormonal iodine released from the thyroid during chronic iodide supplementation and to observe the change of the release rate of such compounds in response to acute or chronic iodide administration. In the thyroid venous plasma of dogs maintained on a regular diet or a diet supplemented with 1 mg of iodide/ day, iodothyronines, iodide and nonhormonal PBI, but not iodotyrosines, were found. However, nonhormonal PBI present in plasma was not released from the thyroid and the nature of nonhormonal iodine released from the thyroid in response to chronic iodide supplements was suggested to be iodide. In both dietary groups of dogs, absolute iodine uptake (AIU) was significantly greater than the net uptake of iodide, suggesting that iodide was actually released from the thyroid. As serum iodide was increased bychronic iodide supplements, AIU increased but the net uptake ...

In 1919 Atwell showed that extracts from mammalian pituitary glands darkened the skin of hypophysectomized tadpoles. Since then the active principle, considered a hormone, has been known as intermedin, melanophore dilating principle, melanophore hormone, etc., with reference to its source in the intermediate lobe of the pituitary gland or its site of action, the melanophores of amphibia. Because the term melanocyte (Fitzpatrick and Lerner, 1953, Gordon, 1953) recently was adopted as the name for the melanin forming cell in all animals, and because mammalian as well as amphibian melanocytes are affected by the pigment-cell activating principle of the pituitary gland, the latter has been designated as melanocyte stimulating hormone (MSH). Most of the early methods for determining MSH consisted of observing in vivo darkening of frogs or fish injected with the hormone. Recently Friedin, Fishbein, and Hisaw (1948) described an in vitro assay in which the darkening of isolated frog

Quantitative studies of iodine metabolism were carried out in iodide-treated hyperthyroid patients in order to observe the alterations of iodine metabolism which occur in response to clinical improvement and to the recurrence of symptoms. Twelve hyperthyroid patients were given 10 mg of iodide as KI t.i.d. and several thyroid function tests were performed at intervals of 2 weeks thereafter. Serum PBI and T3-resin sponge uptake decreased to the normal range within 2 weeks in all patients and these normal values continued until recurrence of thyrotoxicity. One, 2 and 24 hr thyroidal 131I uptakes decreased from 57.4 to 22.6%, from 68.0 to 24.4%, and from 75.1 to 19.9%, respectively, within 2 weeks of the iodide treatment. However, these values were considerably higher than those obtained from similarly treated euthyroid subjects (2.6, 2.5 and 3.4%, respectively). Thyroidal absolute iodine uptake (AIU), calculated from thyroidal clearance of 131I multiplied by serum inorganic 127I, increased about 3-...

Canine growth hormone (CGH) from dog pituitary extract has been purified by a procedure involving alcohol precipitation or ammonium sulfate fractionation, and successive chromatographies on Sephadex G-100 and diethylaminoethyl-Sephadex. The purified CGH preparation showed growth hormone (GH) activity approximately 3 times that of the NIHGH-B12 standard as determined by the tibia test. Its elution patterns obtained in the chromatographic analyses using a Sephadex G-100 column usually showed 2 main peaks. These peaks possessed GH activity similar to that of the material applied on the column. Furthermore, the amino acid compositions of these peaks were essentially the same, in that the content of leucine and glutamic acid was characteristically dominant. The minimal molecular weight of the CGH fractions obtained was estimated to be approximately 24,000 ± 1000 by the chromatographic analysis using Sephadex G-100 and 22,700 ± 200 from the amino acid composition, respectively. The experimental results indicate...

Effects of electrical stimulation of the vagus nerve on the content of PB131I in thyroid venous blood were studied in dogs. After stimulating the nodose ganglion on either side, the concentration of the PB131I in thyroid venous blood and the PB131I output from the thyroid increased in all 14 cases. Unilateral nodose ganglion stimulation facilitated the secretion of PB131I bilaterally, the increase on the stimulated side being always more pronounced than the other. The PB13lI response to nervous stimulation was characterized by a prompt increase within 15 min. Effects of stimulation of the vago-accessory myelencephalic rootlets of the vagus nerve were also investigated to ascertain the pathway through which the nervous stimulus was transmitted to the thyroid for secretion of its hormone.The results indicate that regulation of thyroid hormone secretion by the vagus nerve depends mainly on fibers passing through the main stem of the vagus among its original rootlets. A significant increase in thyroid blood f...

To examine the hypothesis that LATS-protector may cause thyroidal hyperfunction in Graves' disease, the effect of LATS-protector on the intracellular colloid droplet formation in human thyroid tissue was studied in vitro. The IgG's from 9 untreated patients with Graves' disease, 4 thyrotoxic patients who were being treated hut were still thyrotoxic, and 4 normal subjects not showing LATS were chosen to study the presence of LATS-protector and its human thyroid stimulating activity. LATS-protector was assayed as described by Adams with slight modifications. LATS-protector was demonstrated in the IgG's of 6 out of 9 untreated patients with Graves' disease. Their IgG significantly inhibited the inactivation of LATS by the thyroid extract, recovering approximately 30% of the LATS-potency. The IgG from the treated, but thyrotoxic patients or from the normal individual did not show such an inhibition. When sliced normal human thyroid tissue was incubated with the IgG from thyrotoxic patients, an obvious elevati...

We investigated the action of a synthetic rat atrial natriuretic factor (ANF) with 28 amino acids on renin secretion in rats. Renin release by kidney cortex slices was determined after 90 min of incubation at 37C. ANF inhibited basal renin release in a dose-related fashion. ANF also decreased cAMP release and increased cGMP release in a dose-dependent manner. Renin release stimulated by 10(-7) M isoproterenol was inhibited by ANF with an ID50 of 5.8 x 10(-8) M. The renin-inhibitory effect was not calcium-dependent. In anesthetized rats, a bolus IV dose of ANF decreased plasma renin activity and cAMP concentration, but increased cGMP concentration. These data suggest that ANF inhibits renin secretion via the direct action on juxtaglomerular cells and that this effect may be partly mediated by the changes in cyclic nucleotide production.

Effects of ACTH and ACTH fragments on immunoreactive corticotropin-releasing factor (I-CRF) release were examined by utilizing rat hypothalamic perifusion system and a rat CRF RIA. ACTH-(1-39) had a dose-related inhibitory effect on I-CRF release. Mean percent inhibition of I-CRF release was 52, 55, 49, 30 and less than 5 percent by ACTH-(1-39), ACTH-(1-24), alpha-MSH and ACTH-(18-39) at 2.2 nM concentrations, respectively. These results suggest the presence of a negative short-loop feedback mechanism, and also that the active core is contained within the ACTH-(1-17) structure.

The response of pituitary adenomas obtained surgically from patients with Cushing's disease of Nelson's syndrome to synthetic ovine corticotropin-releasing factor (CRF), vasopressins, somatostatin-28, dexamethasone, 3-isobutylmethylxanthine or high [K+] was examined in vitro by measuring the amount of pro-opiomelanocortin (POMC)-derived peptides secreted into the culture medium. CRF did not stimulate the secretion of adrenocorticotropin-, beta-endorphin-, or gamma 3-melanotropin-like peptides from the pituitary adenomas at concentrations ranging from 1 x 10(-13) M to 1 x 10(-7) M whereas vasopressins, 3-isobutyrl-methylxanthine and high [K+] increased, while somatostatin-28 and dexamethasone suppressed, the secretion of these POMC-derived peptides. These findings suggest that either the pituitary ACTH-producing tumors have lost their receptors to CRF or their post-receptor mechanism to CRF is not functional.

The concentrations of immunoreactive corticotropin-releasing factor (I-CRF) in human cerebrospinal fluid (CSF) were measured utilizing immunoaffinity chromatography and RIA in patients with no endocrine disease, patients with Cushing's disease, Nelson's syndrome, Sheehan's syndrome, Addison's disease and steroid treated patients. On high performance liquid chromatography, the elution profile and retention time of I-CRF in CSF were not identical with ovine CRF. I-CRF concentrations in CSF from patients with Cushing's disease and Sheehan's syndrome were lower than those from normal subjects, however those from patients with Nelson's syndrome and Addison's disease were within the normal range. I-CRF concentrations in CSF from patients with Cushing's disease returned to normal levels 2-9 months after pituitary adenomectomy. These results suggest that CSF I-CRF concentrations are reduced by increased plasma corticosteroid levels.

The effect of naloxone, a specific antagonist of opioid peptides, on plasma.GH, PRL, and cortisol responses to insulin-induced hypoglycerriia was studied in five healthy male subjects. The iv administration of regular insulin (0.15 U/kg) led to similar degrees of hypoglycemia on control and experimental days. Plasma GH, PRL, and cortisol levels rose significantly in response to insulin-induced hypoglycemia. A 2-h infusion of naloxone (0.8 mg/h) started 30 min before insulin injection did not alter either basal hormone levels or the hormone responses to insulin-induced hypoglycemia. These results suggest that endogenous opioid peptides do not play a major role in GH, PRL, or cortisol secretion induced by insulin hypoglycemia.

Corticotropin-releasing factor (C R F)-like activity, which was first found in the hypothalamus, was detected in the extracts of human placentas obtained at full term from spontaneous deliveries. The molecular size of the placental CRF-like substance is slightly larger than that of adrenocorticotropin. In cultures of rat anterior pituitary cells, the placental CRF-like material shows bioactivity similar to that of the partially purified rat hypothalamic CRF and synthetic ovine C R F on the release of adrenocorticotropin and (β-endorphin. Furthermore, the CRF-like activity from human placenta was attenuated by trypsin digestion. Thus the human placenta may elaborate a substance similar to hypothalamic CRF.

An antiserum raised against human pancreatic growth hormone-releasing factor-(1-40) (hpGRF-40) was found to recognize the rat hypothalamic growth hormone-releasing factor (rhGRF): This antiserum, when given iv to adult male rats, completely abolished GH release induced by a synthetic rhGRF (0.2, 1 microgram/kg BW), and inhibited the physiological GH secretion over 2 days. After passive immunization with anti-hpGRF-40 serum, iv injection of either FK 33-824 (100 micrograms/kg BW), an enkephalin analogue, or clonidine (125 micrograms/kg BW), an alpha-adrenergic agent, failed to stimulate GH release in the unanesthetized rat. These results indicate that the GH-releasing actions of opiatergic and alpha-adrenergic mechanisms are mediated through stimulation of endogenous rhGRF release in the rat.