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Context: -Pituitary adenoma classification is complex, and diagnostic strategies vary greatly from laboratory to laboratory. No optimal diagnostic algorithm has been defined., Objective: -To develop a panel of immunohistochemical (IHC) stains that provides the optimal combination of cost, accuracy, and ease of use., Design: -We examined 136 pituitary adenomas with stains of steroidogenic factor 1 (SF-1), Pit-1, anterior pituitary hormones, cytokeratin CAM5.2, and α subunit of human chorionic gonadotropin. Immunohistochemical staining was scored using the Allred system. Adenomas were assigned to a gold standard class based on IHC results and available clinical and serologic information. Correlation and cluster analyses were used to develop an algorithm for parsimoniously classifying adenomas., Results: -The algorithm entailed a 1- or 2-step process: (1) a screening step consisting of IHC stains for SF-1, Pit-1, and adrenocorticotropic hormone; and (2) when screening IHC pattern and clinical history were not clearly gonadotrophic (SF-1 positive only), corticotrophic (adrenocorticotropic hormone positive only), or IHC null cell (negative-screening IHC), we subsequently used IHC for prolactin, growth hormone, thyroid-stimulating hormone, and cytokeratin CAM5.2., Conclusions: -Comparison between diagnoses generated by our algorithm and the gold standard diagnoses showed excellent agreement. When compared with a commonly used panel using 6 IHC for anterior pituitary hormones plus IHC for a low-molecular-weight cytokeratin in certain tumors, our algorithm uses approximately one-third fewer IHC stains and detects gonadotroph adenomas with greater sensitivity.

Objective: Thyroid-stimulating hormone (TSH)-expressing pituitary adenomas are a rare but important entity with a spectrum of clinical manifestations. There are currently no data to indicate whether a difference exists in the natural progression of active and silent TSH-expressing pituitary adenomas (defined by the presence or absence of clinical hyperthyroidism, respectively). Here we report our experience (including presenting symptoms, treatment, and outcome) with managing both groups over 11 years in the largest single-center study published to date., Methods: We reviewed retrospectively all patients with histopathologically proven TSH-expressing pituitary adenomas who presented to our center between 2002 and 2012. Data reviewed included clinical presentation, biochemical status, tumor size, management, histopathologic results, and long-term postoperative outcomes., Results: A total of 32 patients (16 male) were identified from a total of 902 operations for pituitary adenomas performed between 2002 and 2012. Mean follow-up was 6.7 years. One-quarter (25%) of patients were clinically hyperthyroid at presentation. Visual disturbance was the commonest presenting complaint in 34%. All patients underwent transsphenoidal surgery. Thirty-one percent of patients had a recurrence. The clinically active and silent TSH-expressing pituitary adenomas behaved in a similar manner with respect to recurrence rates., Conclusions: TSH-expressing pituitary adenomas present with a wide clinical spectrum. Visual disturbances are common. Despite radiologic evidence of clearance after surgery and extended follow-up, they may still recur whether clinically "active" or "silent." Our data support the need for close, long-term follow-up of these patients., (Copyright © 2014 Elsevier Inc. All rights reserved.)

While TSH-producing adenoma (TSHoma) is rare, the diagnosis is often delayed because the clinical features are heterogeneous. The patient was a 69-year-old woman who had been referred to the Yachiyo Medical Center in August 2008, because of dyspnea, loss of appetite, weight loss of 10 kg, and diarrhea that lasted 4 years. We diagnosed this patient with pituitary TSH-producing macroadenoma. Thyroid hormone concentration was increasing although the serum TSH level was within a normal range after trans-sphenoidal surgery. We considered that because of enlargement of the thyroid gland due to long-term stimulation by TSH, a low concentration of TSH could stimulate the thyroid gland to produce excess T3 or T4. The somatostatin analogue, octreotide was used to control the TSHoma and serum TSH concentration but not thyroid hormone. The octreotide in combination with thiamazole treatment for 14 months controlled thyroid hormone concentration and decreased the thyroid mass, and ultimately, the thiamazole could be stopped. To date, the use of combination therapy of octreotide with thiamazole in patients with remaining TSH-producing adenoma without Basedow's disease is rare, and we suggest that this treatment is one of the therapeutic means to treat recurrence of TSH-producing adenoma after surgery with progressive complications or large thyroid gland.

Background: Endocrine gland-derived vascular endothelial growth factor (EG-VEGF) is an angiogenic molecule restricted to endocrine glands and, particularly, to steroid-secreting cells. The expression of EG-VEGF and its significance in human adenohypophysis in physiological and pathological conditions is still unknown., Materials and Methods: In this study, we investigated by immunohistochemistry the expression of EG-VEGF in 2 samples of normal adenohypophysis and 43 bioptic samples of pituitary adenoma. Moreover, the expression of growth hormone (GH), prolactin (PRL), follicle-stimulating hormone (FSH), luteinizing hormone (LH), thyroid-stimulating hormone (TSH) and adrenocorticoprophic hormone (ACTH) were also estimated., Results: The results of this study for the first time demonstrate a down-regulation of EG-VEGF expression in human pituitary adenoma as compared to normal adenohypophysis, suggesting an impaired function of the neoplastic cells in terms of hormone release in the blood stream, as a consequence of impaired tumor angiogenesis in the tumor., Conclusion: On the basis of our data showing a marked decrease in the expression of EG-VEGF in pituitary adenoma, with the exception of LH-secreting adenomas, we suggest that LH might be involved in the induction of EG-VEGF secretion.

Background: A thyrotropin (TSH)-secreting pituitary adenoma coexisting with differentiated thyroid carcinoma is rare. There have been only four previously reported cases; three were treated with thyroidectomy followed by pituitary resection and one was treated with thyroidectomy alone., Methods: We hereby report the fifth case, in which a patient presented with a TSH/growth-hormone-secreting pituitary macroadenoma coexisting with papillary thyroid carcinoma (PTC)., Results: She underwent biochemical testing, ophthalmologic examination, thyroid ultrasonography, Tc-99m-pertechnetate thyroid scan, whole-body positron emission tomography, (111)In-octreotide scan, thyroid fine-needle aspiration biopsy, octreotide treatment, total thyroidectomy, recombinant human TSH radioactive iodine remnant ablation, and continued treatment with octreotide and levothyroxine after thyroidectomy. She has remained asymptomatic for 24 months without biochemical or radiological evidence of pituitary hormone oversecretion, pituitary adenoma enlargement, and PTC recurrence., Conclusion: To our knowledge, this is the first case of a TSH/growth-hormone-secreting pituitary macroadenoma coexisting with PTC being successfully treated with octreotide and levothyroxine after thyroidectomy and recombinant human TSH-stimulated radioactive iodine remnant ablation.

Thyrotropin (thyroid-stimulating hormone (TSH))-producing pituitary adenomas have been known to be quite variable in clinical features covering from typical functioning TSH-producing adenomas (FTSHomas) associated with hyperthyroidism to clinically silent TSH cell adenomas (STAs) that are apparently unassociated with hyperthyroidism. It is important to distinguish STAs from other types of clinically non-functioning adenomas for adequate postoperative managements. However, because of rareness of TSH-producing adenomas, their histopathological features linking to the clinical manifestations have not been well characterized. Herein, we investigated clinical and histopathological findings to characterize 29 TSH-producing adenomas including 20 FTSHomas and nine STAs. Clinical symptoms of the patients with STAs included headache, visual defect, vertigo, and nausea. All STAs and 19 FTSHomas were macroadenoma. The average tumor size of STAs was significantly larger than that of FTSHomas (P < 0.05). The invasiveness was detected in 33% STAs and in 20% FTSHomas. Both STAs and FTSHomas showed a variety of morphological features and immunohistochemical profiles. Chromophobic polygonal or short-spindled tumor cells usually proliferated in a diffuse pattern, while they exhibited globoid or whorl-like appearance with intertwined cytoplasmic processes in both subgroups. Stromal fibrosis and calcification were often noted. Their nuclei were somehow pleomorphic. Ultrastructural features of all four STAs examined were similar to those of normal thyrotrophs. Thus, STAs and FTSHomas were indistinguishable by histology alone. Immunohistochemically, the number of TSH-positive cells in individual FTSHomas was highly various. Six tumors showed only a few TSH-positive cells (1-5%), and three were negative for TSH by conventional method without antigen retrieval. After proteinase K treatment, these tumors turned out TSH positive. As defined, STAs were TSH positive in more than 20% of tumor cells and three of them in more than 50%. Growth hormone- and/or prolactin-positive cells were detected in 55% STAs and 63% FTSHomas. Both pituitary-specific transcription factor 1 and GATA-binding protein 2 were expressed in all STAs and 20 FTSHomas. Membranous somatostatin receptor (SSTR)-2A immunoreactivity was found in 89% STAs and 94% FTSHomas, whereas SSTR5 was positive in 78% of both STAs and FTSHomas. MIB-1 labeling index was related to tumor invasiveness and tumor size (P < 0.05, P = 0.09, respectively). Thus, although both STAs and FTSHomas showed unique histopathological features distinct from other type adenomas, these two subgroups were indistinguishable by histopathology. Immunohistochemistry for TSH by use of antigen retrieval, transcription factors, and SSTRs may be useful to confirm STAs and to determine the postoperative therapy among various kinds of clinically non-functioning adenomas.

Objective and Importance: Pituitary adenomas producing primarily FSH and to a lesser extent GH, LH, alpha-subunit, TSH and PRL without clinical or laboratory evidence of increased hormone release have not previously been reported. Our aim was to obtain some insight into the possible cytogenesis of this unusual tumor., Clinical Presentation: A 65-year-old woman presented with headaches. Magnetic resonance imaging (MRI) demonstrated a sellar mass. Pituitary hormone assays showed normal blood levels. The tumor was removed by the transsphenoidal approach., Result: By light microscopy, the adenoma was chromophobic, weakly PAS-positive, and immunoreactive mainly for FSH (85%) and to a lesser extent for GH (30%), LH (15%), alpha-subunit (3%), TSH (2%), and PRL (1%). Although double immunostaining showed hormone reactivities to be localized largely in separate distinct cells, the tumor was ultrastructurally monomorphous, i.e., consisted of a single-cell type, resembling gonadotrophs., Conclusion: The cytogenesis of plurihormonal pituitary adenomas is not fully understood. Further investigations are required to clarify the basis for their plurihormonality despite an ultrastructural gonadotroph phenotype.

Pituitary carcinomas are exceedingly rare. At present, the sole diagnostic criterion is metastatic spread, either craniospinal or systemic. There is no agreement on a histologic, immunohistochemical, and/or ultrastructural definition. We report a clinically and morphologically well-documented example of pituitary thyrotropin cell carcinoma in a man with multiple endocrine neoplasia type 1 syndrome. The tumor produced thyrotropin, alpha-subunit, and prolactin and, through electron microscopy, was found to consist solely of Thyrotroph cells. Over a protracted course, craniospinal and systemic metastases were noted. The primary and metastatic deposits of this aggressive tumor were studied. To our knowledge, this tumor is the first reported case of thyrotropin cell carcinoma occurring in association with the multiple endocrine neoplasia type 1 syndrome. The literature regarding thyrotropin carcinomas is reviewed. Based on the study of several biopsies during disease progression, we believe that the carcinoma originated de novo without an intermediary adenoma phase.

Pituitary adenomas were identified in 14 of 491 (2.9%) cynomolgus macaques evaluated from 1994 to 2004. Cases included male (8) and female (6) cynomolgus macaques ranging from 18 to 32 years of age. Seven of the pituitary adenomas caused gross enlargement of the pituitary gland that was visible on postmortem examination, whereas the remaining 7 were multifocal microadenomas identified on histologic examination. A total of 35 adenomas were identified in the 14 macaques, 6 of which were being treated for diabetes mellitus. Mean (+/- SD) pituitary weight was 0.31 +/- 0.42 g, compared with 0.07 +/- 0.02 g for 430 historical control animals (P < 0.0001). Immunohistochemical staining for follicle-stimulating hormone, luteinizing hormone, prolactin, human growth hormone, thyroid-stimulating hormone, and adrenocorticotropic hormone was applied to pituitary tissue from all cases. Immunostaining revealed 22 of 35 (62.9%) lactotroph adenomas, 5 of 35 (14.3%) plurihormonal cell adenomas, 3 of 35 (8.6%) corticotroph adenomas, 2 of 35 (5.7%) null cell adenomas, 1 of 35 (2.9%) somatotroph adenomas, 1 of 35 (2.9%) mixed corticotroph-somatotroph adenomas, 1 of 35 (2.9%) mixed lactotroph-corticotroph adenomas, 0 of 35 gonadotroph adenomas, and 0 of 35 thyrotroph adenomas. This study represents the first extensive retrospective case series performed to evaluate the histologic and immunohistochemical characteristics of pituitary adenomas in cynomolgus macaques. Our findings indicated that macaque pituitary adenomas frequently had mixed histologic appearance and hormone expression, and that, similar to human pituitary adenomas, prolactin-secreting neoplasms were the most prevalent type.

Objectives: The examination of potential associations between Graves' disease and thyrotropin-producing pituitary adenoma (TSHoma) after treatment using octreotide, and of the expression of peroxisome proliferator-activated receptor gamma (PPAR gamma)., Design and Methods: A specimen of resected TSHoma tissue from our case was immunohistochemically examined for expression of somatostatin receptor 2A (SSTR2A) and PPAR gamma. Specimens of thyroid tissue from two cases with Hashimoto's thyroiditis were immunohistochemically examined for expression of SSTR2A., Results: Expression of SSTR2A and PPAR gamma was identified in TSHoma cells. SSTR2A was also expressed in lymphocytes that had infiltrated thyroid tissue in Hashimoto's thyroiditis. In previous reports, three of four patients with TSHoma displayed Graves' disease after tumor resection, and TSH is also known to play a major role in regulating immunomodulatory gene expression in thyrocytes., Conclusions: Both the immunomodulatory effects of octreotide on intrathyroidal lymphocytes and rapid reductions in TSH may contribute to the onset of Graves' disease. Patients with TSHoma-associated autoimmune thyroiditis should undergo careful follow-up for development of Graves' disease after treatment. Both octreotide and the PPAR gamma receptor-activating ligands, thiazolidinediones, may be effective for patients with TSHoma.

We reported a rare case of pleomorphic TSH-producing pituitary adenoma with calcification and reviewed the literature. A 25-year-old female was admitted to our hospital with a complaint of anterior neck swelling. An endocrinological examination demonstrated elevated serum levels of free triiodothyronine (FT3: 5.6 pg/ml), free thyroxin (FT4: 2.2 ng/dl), TSH (5.85 microIU/ml), and TSH a-subunit (5.9 ng/ml), whereas a physical examination revealed no goiter. CT scan showed a suprasellar isodensity mass with dense calcification. Contrast-enhanced T1-weighted images revealed a less enhancing tumor extending from the left upper portion of pituitary fossa into suprasellar cistern. The patient underwent gross total removal of the tumor via the right pterional approach. Microscopically, medium-sized to enlarged tumor cells with marked pleomorphism and prominent calcification were observed. The tumor cells displayed positive reaction for TSH (beta-subunit). The MIB-1 index averaged 2.9%. The histological diagnosis was a pleomorphic TSH-producing pituitary adenoma. Postoperatively, the serum levels of FT3, FT4, TSH, and TSH alpha-subunit decreased to normal range. Follow-up MR images showed no evidence of recurrent tumor 3 years after the resection. All of six patients with densely calcified TSH-producing pituitary adenoma, previously reported in the literature, remained well without tumor recurrence. We suggest that this type of TSH-producing pituitary adenoma may be associated with favorable prognosis despite histologically pleomorphic appearance.

Objective: A clinicopathological and clonal study of a pituitary tumor was made in a 26-year-old woman with chronic thyroiditis to differentiate TSH-producing adenoma from TSH hyperplasia., Methods: Tumor specimens were subjected to histopathological study and clonal analysis (HUMARA)., Results: Immunohistochemical examination disclosed TSH-beta, PRL, GH, ACTH, FSH-beta, LH-beta, and alpha-subunit production in the adenoma cells. These heterogeneous phenotypes are characteristic of both thyrotroph hyperplasia and plurihormonal TSH-producing adenoma. However, the HUMARA method demonstrated monoclonality of the tumor cells., Conclusion: Monoclonality of the tumor cells proved that the pituitary tumor was plurihormonal TSH-producing adenoma, not TSH hyperplasia., (Copyright 2003 S. Karger AG, Basel)

Objective: Local 5'-deiodination of l-thyroxine (T(4)) to active thyroid hormone 3,3',5-tri-iodothyronine (T(3)) catalyzed by the two 5'-deiodinase enzymes (D1 and D2) regulates various T(3)-dependent functions in the anterior pituitary and has been well studied in rodents. Only limited information about deiodinase expression and its cellular distribution in human anterior pituitaries is available., Design: We examined 5'-deiodinase enzyme activities in pituitary adenomas (18 non-functioning, seven TSH-producing, one GH- and TSH-producing, five GH-producing, eight prolactin (PRL)-producing, two adenomas each from patients with Cushing's disease and Nelson's syndrome) and three normal anterior pituitaries., Methods: Activities were measured as release of (125)I(-) from tyrosyl-ring labeled reverse T(3) with or without propylthiouracil, a potent inhibitor of D1 which does not influence D2 activities., Results: Most of the adenomas and normal tissues expressed both isoenzymes, with D2 activity higher than D1. In a few tissues D1 activity was higher than D2 and some tissues did not express D1 activity at all. Highest activities of both enzymes were found in TSH- and PRL-producing adenomas but absolute activities and the D1/D2 ratio were variable in the same kind of tumor in different patients., Conclusion: The finding that all examined tissues expressed 5'-deiodinase activity, most of them expressing both isoenzymes, implies that both enzymes are still active in tumors and that local deiodination is important for the function and feedback regulation of human anterior pituitary.

We report a very rare case of thyrotropin (thyroxin stimulating hormone, TSH)-producing pituitary adenoma coexisting with a papillary adenocarcinoma of the thyroid. A 45-year-old woman presented with hyperhidrosis and a nodule in the left thyroid that was first noticed one year earlier. An endocrinological examination showed elevated serum levels of free triiodothyronine (T3) and free throxin (T4) without inhibition of TSH, suggesting the presence of syndromes of inappropriate secretion of TSH. A specimen obtained by needle aspiration of the thyroid nodule revealed the presence of papillary adenocarcinoma. Magnetic resonance images demonstrated a pituitary macroadenoma. The patient was diagnosed as having a TSH-producing pituitary adenoma coexisting with a papillary adenocarcinoma of the thyroid. The patient underwent a total thyroidectomy with resection of the neighboring lymph nodes. Two weeks after this surgery, the pituitary adenoma was totally removed via a pterional approach. Histological and immunohistochemical examinations of the surgical specimens confirmed the lesion as a papillary adenocarcinoma of the thyroid and a TSH-producing pituitary adenoma. Serum TSH levels decreased to undetectable levels immediately after the surgery for the pituitary adenoma. Prolonged stimulation of the thyroid gland by TSH may be involved in the growth of thyroid carcinoma. In cases with a TSH-producing pituitary adenoma, the possible coexistence of thyroid carcinoma should be carefully ruled out. In such cases, a total thyroidectomy followed by TSH level normalization should be performed. Incomplete removal of the thyroid might enable the carcinoma to re-grow if TSH level can not be normalized after the pituitary adenomectomy.

We investigated propofol pharmacokinetics in a patient with secondary hyperthyroidism caused by thyroid stimulating hormone (TSH) producing pituitary adenoma. Laboratory data indicated thyrotoxic state with elevated TSH, FT3 and FT4 levels. General anesthesia was maintained with a doubled propofol infusion rate (8-10 mg.kg-1.hr-1) compared to our standard procedure. During 370 min of infusion, propofol concentrations in arterial blood were kept within optimal ranges (2-4 micrograms.ml-1). Although the clearance of propofol was high (2.8 l.min-1) because of the thyrotoxic state, the patient showed delayed recovery from anesthesia. The half-life of blood propofol after the termination of infusion exceeded 30 minutes (normal: 10-15 minutes). We conclude that the delayed recovery was due to the accumulation of propofol in the adipose tissue during long-term infusion in spite of the increased propofol clearance.

A 27-year-old woman who presented with a left thyroid nodule was found to have hyperthyroidism caused by a syndrome of inappropriate secretion of TSH. The levels of free T3, free T4 and TSH were 9.50 pg/mL, 4.05 ng/dL and 2.16 microU/mL, respectively. Magnetic resonance imaging of the head revealed a pituitary macroadenoma. The TSH response to TRH stimulation was normal and responses of other anterior pituitary hormones to stimulation tests were also normally preserved. Administration of octreotide with iodine successfully reversed hyperthyroidism prior to total resection of pituitary adenoma, which was followed by hemithyroidectomy of the left thyroid five months later. Histologically, the resected pituitary adenoma was a TSH-producing adenoma (TSH-oma) and the thyroid nodule was a papillary adenocarcinoma. Serum TSH diminished to undetectable levels immediately following pituitary adenomectomy but gradually normalized over nine months. Coexistence of a TSH-oma with thyroid cancer is very rare and only two similar cases have previously been documented. This combination raises the possibility that TSH may be involved in tumorigenesis in the thyroid gland.

Thyroid hormone inhibits thyrotropin (TSH) production and thyrotrope growth. Somatostatin has been implicated as a synergistic factor in the inhibition of thyrotrope function. We have previously shown that pharmacological doses of thyroid hormone (levothyroxine [LT4]) inhibit growth of murine TtT-97 thyrotropic tumors in association with upregulation of somatostatin receptor type 5 (sst5) mRNA and somatostatin receptor binding. In the current study, we examined the effect of physiological thyroid hormone replacement alone or in combination with the long-acting somatostatin analogue, Sandostatin LAR, on thyrotropic tumor growth, thyrotropin growth factor-beta (TSH-beta), and sst5 mRNA expression, as well as somatostatin receptor binding sites. Physiological LT4 replacement therapy resulted in tumor shrinkage in association with increased sst5 mRNA levels, reduced TSH-beta mRNA levels and enhanced somatostatin receptor binding. Sandostatin LAR alone had no effect on any parameter measured. However, Sandostatin LAR combined with LT4 synergistically inhibited TSH-beta mRNA production and reduced final tumor weights to a greater degree. In this paradigm, Sandostatin LAR required a euthyroid status to alter thyrotrope parameters. These data suggest an important interaction between the somatostatinergic system and thyroid hormone in the regulation of thyrotrope cell structure and function.

Many malignancies may present with metastases to the pituitary gland. The association of thyroid carcinoma with pituitary metastases is, however, very rare. This report describes two patients in whom metastases from a papillary thyroid carcinoma to the pituitary gland resulted in panhypopituitarism with blunted endogenous thyrotropin (TSH) production following withdrawal of levothyroxine. Both required the use of recombinant human TSH prior to radioiodine therapy. Symptoms of hypopituitarism may be difficult to distinguish clinically from those of hypothyroidism in the setting of levothyroxine withdrawal. Clinicians should be aware of the clinical and biochemical manifestations of this rare association.

We present the case of a 7-month-old baby with Cushing's disease due to an adrenocorticotropic hormone (ACTH)-secreting pituitary adenoma combined with cells producing thyreotropin-secreting hormone (TSH). In MRI scans, a contrast-enhancing lesion was seen inside the pituitary fossa, and it extended into the suprasellar region. On the assumption of a pituitary adenoma, surgery was performed. Corresponding with biochemical findings, histopathological evaluation revealed an ACTH- and TSH-producing tumor. Genetic analysis did not demonstrate an alteration at codon 201 (Arg) and 227 (Glu). To our knowledge, this is the first case described in a child of this age.

Two of 420 patients with pituitary adenoma who underwent operation from 1989 to 1997 had thyroid stimulating hormone (TSH) producing adenoma. We investigated these TSH cell adenomas with immunohistochemical and ultrastructural methods and compared their ultrastructural features with brefeldin A (BFA, 0.5 mg/ml) treated pituitary adenoma cells. BFA-treated pituitary adenomas include a prolactin (PRL) cell adenoma, a growth hormone (GH) cell adenoma, an adrenocorticotropic hormone (ACTH) cell adenoma, a gonadotroph adenoma, and a plurihormonal adenoma. Immunohistochemical staining disclosed that one of the TSH cell adenomas produced only TSH-beta and that another produces both TSH-beta and FSH-beta. Ultrastructural analysis showed the abundance of oval-shaped dilated rough endoplasmic reticulum (rER). Within the dilated rER, the mistlike deposit or deposit along the inner margin of the rER membrane was observed. On the other hand, BFA-treated cultured pituitary adenoma cells showed the opening of the cavity of the rER cisterna and they enlarged to an oval form with time and revealed an accumulation of electron-dense deposits within the dilated rER. These ultrastructural similarities between TSH cell adenoma and BFA-treated pituitary adenoma cells indicate the functional disturbances in the secretory passage through the Golgi apparatus in TSH cell adenoma cells.

The pathologist plays an important role in the distinction of pituitary adenomas from other tumors and tumor-like lesions of the sellar region, and in the accurate morphologic characterization of pitutiary adenomas. A clinicopathologic classification of pituitary adenomas is based on cell differentiation correlated with clinical evidence of hormone secretion; this classification emphasizes clinically relevant features that can offer guidance for patient management. The application of a rational approach to the immunohistochemical analysis of these lesions can be used to evaluate pathogenetic and prognostic markers and to predict responses to specific therapeutic modalities.

Elevated levels of free triiodothyronine (fT3) of 8.8 ng/dl (normal range 2.0 to 4.2) and free thyroxin (fT4) of 3.5 pg/ml (0.8 to 1.7) were found in the course of an examination of a 53-year old patient due to a planned hysterectomy. As thyrotropin (TSH) also was elevated with 5.8 mU/l (0.4 to 4.5), these findings corresponded to an inappropriate secretion of TSH (IST). Additional examinations revealed a blunted rise of TSH secretion after i.v. injection of 200 micrograms thyrotropin releasing hormone (TRH) as well as lacking suppression of TSH secretion after oral doses of 75 micrograms T3 during one week. alpha-TSH levels with 3.7 micrograms/l were elevated in comparison to a matched normal sample just as the molar ratio alpha-TSH/TSH with 6.95 and sex hormone-binding globulin (SHBG) with 175 nmol/l and showed an absence of inhibition in the T3 suppression test. These results were suggestive of neoplastic inappropriate secretion of TSH (nIST) due to a TSH-secreting pituitary adenoma. In concordance, the magnetic resonance imaging (MRI) showed a 1 cm tumor in the sella. The adenoma could also be visualized by 111In-octreotide and 123I-epidepride scintigraphies of the pituitary gland. After transsphenoidal resection, histological examination of the tumor resulted in the finding of a TSH-secreting adenoma. Postoperative TSH levels were not detectable, indicating the complete removal of the adenoma. Levels of fT3 and fT4 were slightly below normal with 1.9 pg/ml and 0.7 ng/dl, respectively. A control scintigraphy with 111In-octreotide following an equivocal MRI showed no uptake in the pituitary.

Thyrotropin (TSH)-secreting pituitary adenomas cause hyperthyroxinemia in the presence of "inappropriately" elevated concentrations of TSH. TSH production under these circumstances escapes the normal negative feedback effect of thyroid hormone. We propose that this defective negative feedback is mediated by an abnormality of thyroid hormone receptor (TR) expression. Two TSH-secreting pituitary adenomas were analyzed by immunocytochemistry for TR isoform protein expression and by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) for TR isoform mRNA expression. The results obtained from these tumors were compared with the findings from six normal human pituitaries. Neither tumor examined expressed detectable levels of nuclear TRalpha or TRbeta proteins, in contrast to the normal pituitaries studied, which expressed all TR isoforms. Application of RT-PCR, however, revealed mRNAs encoding each TR isoform in all tumorous and normal tissues examined. Semiquantitative RT-PCR revealed similar levels of expression of TRalpha and TRbeta isoform mRNAs in tumors and normal tissue, in contrast to the observed difference in TR proteins. Absent TRalpha and TRbeta protein expression, in association with normal mRNA levels, implies a post-transcriptional defect in TR mRNA processing in TSH-secreting adenomas. Reduced TR expression in these tumors may explain defective negative feedback of thyroid hormone on TSH production, and may also contribute to uncontrolled tumor growth.

The GH4C1 pituitary cell line, an excellent model for a thyroid hormone action study, was used for determination of the relationship between thyroid hormone receptor occupancy and intensity of cell proliferation, prolactin (PRL) production, thyrotropin (TSH) inhibition and 3,5,3,-L-triiodothyronine (T3) receptor down-regulation. Nuclear receptor population was progressively occupied by T3 in concentrations ranging from 0.025 to 10.0 nM T3. Bmax ranged from 0.029 fmol/10(6) cells at the lowest T3 concentration to Bmax = 12.51 fmol/10(6) cells at the highest concentration. Each of the observed biological events is operative within distinct dose-response ranges in cultured GH4C1 cells. The maximal biological response (except the TSH inhibition and T3 receptor down-regulation) does not require the occupation of the whole nuclear receptor population by T3 and the intensity of none of the responses studied was directly proportional to thyroid hormone receptor occupancy.

Incubation media of pituitary cells and homogenates of pituitary tumors were studied by isoelectric focusing followed by radioimmunoassay. Almost all LH-immunoreactive components detected had been observed in earlier studies on highly purified preparations. In one pituitary tumor homogenate, intact LH (LHi) was detected. The majority of LHi-components present in this tumor were relatively acidic (LH Type I) components. The corresponding beta-subunit population was in agreement with the one earlier observed for Type I LH. In all other tumor homogenates only alpha- and beta-subunits could be detected. In incubation media of pituitary cells from patients with no known endocrine disease nor a pituitary tumor highly acidic populations of alpha-subunits were detected. In only one of the incubation media significant amounts of LHi and free beta-subunits were observed. In an incubation medium of a TSH-producing pituitary tumor significant amounts of intact LH, free alpha- and beta-subunits were detected. The cell contents of this tumor contained components with lower pl-values than the medium. In general a good correlation is observed between the population of LH-components in material from individual identifiable pituitary glands and in highly purified preparations from large numbers of anonymous pituitary glands.

Seventy-five clinically non-functioning pituitary adenomas were characterized in terms of their histologic and immunohistochemical features. Fourteen adenomas (18.7%) were positive for hormones other than gonadotropins. These included two somatotroph adenomas, three lactotroph adenomas, four thyrotroph adenomas, four corticotroph adenomas and one unusual plurihormonal adenoma. Fifty-five adenomas (73.3%) were exclusively positive for one or more gonadotropin subunits (beta-follicle stimulating hormone, beta-luteinizing hormone, and alpha-subunit of glycoprotein hormones), but negative for other hormones such as growth hormone and beta-thyrotropin. Histologically, a papillary arrangement along the capillary was most characteristically observed in the gonadotropin-positive adenomas. Five of six adenomas negative for any pituitary hormones exhibited the typical papillary structure. Thus, approximately 80% of clinically non-functioning adenomas constituted a single tumor group that shared the common histologic features of gonadotroph adenomas. These findings suggest that nearly all tumor types of clinically non-functioning adenomas can be diagnosed solely by light microscopy in combination with immunohistochemistry, and that the vast majority of them may be gonadotroph adenomas.

To evaluate the biology of thyrotropin (TSH)-producing pituitary adenomas, the authors reviewed the charts of 19 patients who underwent transsphenoidal surgery within a 15-year period at the University of California, San Francisco (UCSF). Between 1989 and 1991, the period during which immunostaining techniques were used consistently for diagnosis, 2.8% of the pituitary adenomas treated at UCSF were TSH-producing. The rate of reoperation for tumor recurrence was 10.5%. Before pituitary surgery, more than one-third of the 19 patients had undergone thyroid ablation. Two patients had a history of Hashimoto's thyroiditis. The female:male ratio was 1.7:1. Women tended to develop these tumors at a younger age and had a longer history of symptoms but their tumors were smaller and less often invasive than those seen in men. About 50% of the tumors were purely TSH-producing and 50% were plurihormonal, including five that produced both TSH and adrenocorticotroph hormone. All tumors were macroadenomas. Before surgery, 46% of the patients had abnormal electrocardiographic findings; 16% had a rapid onset of severe neurological conditions either before or after surgery. It is concluded that TSH-producing adenomas are more common in patients who undergo surgical treatment than was previously thought. In addition, they occur more frequently in women, have a different biology in women than in men, and tend to be associated with potentially life-threatening cardiovascular and neurological complications.

A 21-yr-old female with hyperthyroidism is described. Though her serum-free T3 was 17.8 pmol/L and free T4 was 60.2 pmol/L, TSH was as high as 10.7 mU/L. TRH stimulated an increase in TSH from 10.7-91.7 mU/L. T3 administration in gradually increasing doses of 100, 200, and 400 mg/day resulted in gradual reduction in serum TSH. Cranial computed tomography and magnetic resonance imaging revealed a microadenoma of the pituitary gland. Histology of the surgical specimen showed a TSH-producing adenoma with TSH cell cluster islets and decreased numbers of TSH cells in the nonneoplastic pituitary. Cultured cells from the adenoma secreted TSH spontaneously and in response to TRH. This TRH-stimulated TSH secretion was suppressed by T3 in a dose-dependent manner. One year postoperatively, neither residual tumor nor recurrence were seen by computed tomography and magnetic resonance imaging. However TSH, as well as free T3 or T4, was still high and overresponsive to TRH.

Thyrotropic tumors (TtT97) contain mRNA transcripts for three T3-receptor (TR) isoforms, alpha 1, beta 1 and beta 2, and a non-receptor alpha 2-variant. We administered T4 (5 mg/l of drinking water) for one month to TtT97-bearing mice, to examine its effect on tumor growth and tumor TR isoform steady-state mRNA levels. Baseline mice were killed at the start of the experiment, and placebo mice were maintained hypothyroid. The treated tumors were 30-35% smaller than the baseline tumors (p = NS), while the placebo tumors were 2- to 7-fold larger than the baseline tumors (p < 0.05). TR beta 1 mRNA increased 5- to 6-fold, while TR beta 2 mRNA decreased by 76%. TR alpha 1 and the alpha 2-variant decreased by 52% and 70%, respectively. Therefore, the tumors decreased their growth rate in response to T4 administration, and increased the ratio of TR beta 1 to TR beta 2 mRNA. This raises the intriguing possibility of a correlation between the relative abundance of the TR beta isoforms and tumor growth.

A 50 year old man with hyperthyroidism secondary to inappropriate secretion of TSH is described. On presentation T3 (42.1 nmol/L), T4 (265 nmol/L) and TSH (17.9 mU/L) were all markedly elevated. A diagnosis of a TSH-secreting pituitary tumor was suspected on the basis of a blunted TSH response to TRH and the absence of suppression of TSH by T3 or bromocriptine, but TSH/alpha subunit molar ratios were uncharacteristically less than 1. Nevertheless, the presence of a tumor was confirmed by computed tomography which demonstrated a 15 mm pituitary macroadenoma. The patient was treated with octreotide which resulted in normalisation of thyroid hormone levels. The duration of action of a single 100 micrograms injection of octreotide was at least 56 hours. The suppression of thyroid hormone levels was similar regardless of the treatment regimen with octreotide (100 micrograms tid, 250 micrograms bid, 100 micrograms bid and continuous subcutaneous infusion (CSI] and no escape was observed during a 16 month treatment period. TSH alpha subunit concentrations were also suppressed during long-term treatment with octreotide (3.3 micrograms/L falling to 1.1 micrograms/L), although no acute changes were noted after administration of single dose octreotide 100 micrograms. Three times the octreotide therapy was discontinued. The incremental rise in TSH and the maximum level of TSH achieved was less on each subsequent occasion, suggesting a suppressive effect of octreotide on the tumor itself. Despite suppression of TSH with octreotide over a 13 month period the pituitary tumor showed no shrinkage on repeat MRI scanning. In conclusion, this patient demonstrates that the differential diagnosis of inappropriate TSH secretion based only on biochemical test may be unreliable.(ABSTRACT TRUNCATED AT 250 WORDS)

To determine whether incubation of mouse thyrotropic tissue with TRH in vitro influenced the oligosaccharide structure of TSH, thyrotropic tumor tissue or pituitary tissue was incubated in vitro with [3H]mannose or with [35S]sulfate and [3H]methionine, in the absence or presence of TRH for times up to 24 h. [3H]mannose-labeled oligosaccharides from intracellular TSH and free alpha-subunits were analyzed by paper chromatography, and were predominantly Man9GlcNAc and Man8GlcNAc units both in the absence and presence of TRH. The [35S]sulfate/[3H]methionine ratio in secreted molecules was greater for TSH than for free alpha-subunits; within TSH heterodimers the ratio was greater for beta-subunits than alpha-subunits. The [35S]/[3H] ratio was not altered in TSH or free alpha-subunits by TRH. Analyses of [3H]mannose-labeled charged oligosaccharides by HPLC anion-exchange chromatography revealed similar types of oligosaccharides present on TSH subunits and free alpha-subunits (having one or two sulfate residues, one or two sialic acid residues, or both a sulfate and a sialic acid residue). These charged oligosaccharides occurred in different proportions on TSH subunits compared to free alpha-subunits, and also differed depending on whether the tissue source was tumorous or nontumorous. The proportions of oligosaccharide unit types were not altered by TRH. Thus, while this study provided information concerning the high-mannose and complex oligosaccharides of mouse TSH, there was no evidence that short incubations of tissues with TRH in vitro caused modulation of TSH oligosaccharide structures.

This case report describes a 38-year-old male who was hospitalized for further clarification of clinically mild hyperthyroidism. His increased total hormone levels, the elevated free thyroid hormones and the elevated basal TSH with blunted response to TRH strongly suggested a pituitary adenoma with inappropriate TSH incretion. Transmission computed tomography showed an intrasellar expansion, 16 mm in diameter. The neoplastic TSH production was confirmed by an elevated alpha-subunit and a raised molar alpha-sub/TSH ratio. However, T4 distribution on prealbumin (PA, TTR), albumin (A) and thyroxine binding globulin (TBG) showed a clearly increased binding to PA (39%), indicating additional prealbumin-associated hyperthyroxinemia. The absolute values of PA, A and TBG were within the normal range. After removal of the TSH-producing adenoma, basal TSH, the free thyroid hormones and T4 binding to prealbumin returned to normal. Therefore, the prealbumin-associated hyperthyroxinemia had to be interpreted as a transitory phenomenon related to secondary hyperthyroidism (T4 shift from thyroxine binding globulin to prealbumin) rather than a genetically conditioned anomaly of protein binding.

Thyroid-stimulating hormone (TSH) subunit glycosylation was compared to that of total cell glycoproteins in mouse thyrotropic tumors. Lipid-linked oligosaccharides, total cell glycoproteins, and TSH subunits were labeled with either [3H]mannose, [3H]galactose, or [3H]glucose in pulse and pulse-chase experiments. The various oligosaccharides were isolated respectively by lipid extraction and mild acid hydrolysis, by selective immunoprecipitation, or by acid precipitation followed by trypsin and endoglycosidase H treatment. The nature of the oligosaccharides was assessed by their migration in paper chromatography, their relative incorporation of different precursors, and also their resistance to alpha-mannosidase. At 60 min, lipid-linked oligosaccharides were found to be composed of Glc3-2Man9GlcNAc2, Man9-8GlcNAc2, and Man5GlcNAc2. At 10 or 60 min of labeling, total cell proteins contained Glc3Man9GlcNAc2, Glc1Man9GlcNAc2, Man9GlcNAc2, Glc1Man8GlcNAc2, Man8GlcNAc2, and Man7GlcNAc2. The largest oligosaccharide, Glc3Man9GlcNAc2, had an unusually long half-life of about 2 h. In contrast, no Glc3Man9GlcNAc2 was found either on TSH + alpha subunits or on free beta subunits isolated either by immunoprecipitation or by sodium dodecyl sulfate gel electrophoresis. Instead, primarily Man9GlcNAc2 was found after a 10-min pulse both on TSH + alpha subunits and on beta subunits. When the pulse was followed by a chase up to 2 h, there was a progressive increase in Man8GlcNAc2 in higher amounts on TSH + alpha-subunit carbohydrate chains than on beta subunits.(ABSTRACT TRUNCATED AT 250 WORDS)

Primary cultures from three serially transplanted mouse pituitary thyrotropic tumors, Furth 97A, 97B, and 97C, and one newly generated tumor, NIH 101A, were shown to secrete free alpha subunits and a beta subunit (TSH-beta) in addition to intact TSH. The three glycopeptides were measured in specific, heterologous radioimmunoassays. Basal secretion of alpha subunit was found to be in excess in all four thyrotropic tumor cultures, ranging from 5.7- to 8.9-fold higher than that of intact TSH and 30- to 35-fold higher than that of free TSH-beta. In cultures from 97A, TRH (10(-7) M) stimulated the secretion of TSH, alpha subunit, and TSH-beta by 175-185% and (T4, 10(-5) M) inhibited TSH to 19%, alpha subunit to 68%, and TSH-beta to 58% of control. Secretion of TSH and its subunits in cultures from 97B was not consistently affected by TRH or T4. In cultures from 97C, TSH and alpha secretion was inhibited in a dose-dependent fashion by T3 from 2.5 x10(-9) to 1.6x10(-7) M. Gel chromatography of incubation media from tumor 97A revealed elution volumes of TSH and alpha similar to those of purified rat glycopeptides and confirmed the excess secretion of free alpha subunit. In contrast, the alpha to TSH ratios in media from cultures of pituitary cells from normal and thyroidectomized rats were 1.6 and 0.8, respectively. The total production of TSH and its subunits was measured in cultures from tumor 97A and confirmed that alpha subunit was synthesized excessively. The ratio of the plasma concentrations of alpha to TSH in tumor-bearing mice was 1.6 compared to 1.3 in thyroidectomized mice, and 0.36 in thyroidectomized rats. The alpha to TSH ratios in extracts of the corresponding thyrotropic tissues (tumor or pituitary) were 2.7, 1.7, and 0.40, respectively. These data are analogous to those reported in humans with thyrotropic and certain other pituitary tumors, and support hypothesis that the subunits of glycoprotein hormones are independently synthesized.

Mouse thyrotropic tumor cells grown in primary culture were shown to synthesize TSH and proteins, as determined by the incorporation of radioactive proline into immunoprecipitable TSH and trichloroacetic acid-precipitable proteins. The net TSH content of the cells and medium determined by RIA is also increased during 24 h of incubation, and newly formed hormone is detected in the medium within 1 h after the addition of proline tracer. To study the effect of T4 and T3 on TSH synthesis, cultures were pulse-labeled with [3H]proline after they had been exposed to either T3 or T4. After 48 but not 24 h, exposure to either T3 or T4 was followed by inhibition. When studied after 48 h of incubation, T4, (10(-13) M) or T3 (10(-11) M) at the lowest concentration tested, was inhibitory to TSH synthesis. At concentrations of T4 and T3 greater than 10(-9) M, the inhibitory effects on TSH synthesis were partially reversed, suggesting a biphasic response. Incubation in TRH (10(-7) M) for 24 h led to a significant increase in TSH synthesis, total protein, acid-precipitable protein, and total DNA. The effect of TRH on TSH biosynthesis was a function of the logarithm of its concentration over the range of 10(-11)-10(-7) M. The inhibitory action of 10(-6) M T3 on TSH synthesis was reversed by exposure to 10(-10) or 10(-7) M TRH.

Mouse pituitary thyrotropic tumor minces were labeled with [35S]methionine and fractionated into rough microsomes, intermediate, and low density smooth microsomes. Thyroid-stimulating hormone subunits were mainly in rough microsomes after a 10-min pulse, but with increasing chase times the proportion in smooth microsomes increased. In rough microsomes, small amounts of an alpha subunit precursor of Mr = 11,000 and larger amounts of an alpha form of Mr = 18,000 were rapidly processed to a form of Mr = 21,000, while small amounts of a beta-subunit precursor of M r = 11,000 were processed to a form of Mr = 18,000. Most of the Mr = 18,000 and Mr = 21,000 subunit forms were converted by endoglycosidase H to forms of Mr = 11,000 to 12,000. Small amounts of endoglycosidase H-resistant forms appeared in low density smooth microsomes after a 30-min chase. Subunit combination was not detected at 10 min; combination was first detected at 20 min and increased progressively to a maximum of 61% of beta in the low density smooth microsomes at 60 min of chase. Although alpha of Mr = 11,000 and 18,000, and beta of Mr - 11,000, were not detected in thyroid-stimulating hormone, both endoglycosidase H-sensitive and -resistant alpha subunit of Mr = 21,000 and beta subunit of Mr = 18,000 were found combined. Thus, the rough endoplasmic reticulum (ER) contains only small amounts of nonglycosylated subunits (Mr = 11,000). The major subunit precursors contain one high mannose oligosaccharide (Mr = 18,000), with a second unit being added onto alpha in the rough ER. Combination of alpha (Mr = 21,000) with beta (Mr = 18,000) begins in the rough ER but occurs predominantly in the smooth ER/Golgi. Oligosaccharides of both combined and uncombined subunits are processed from high mannose to complex forms predominantly in the smooth ER/Golgi.

We have previously demonstrated differences in several cellular responses to TRH in mouse thyrotropic pituitary (TtT) cells and in rat mammotropic pituitary (GH3) cells. In this report, we further explore the mechanism of TRH action in TtT cells by measuring its effects on phosphoinositides and on cytoplasmic free Ca2+ concentration [( Ca2+]i). We demonstrate that TRH stimulates rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] by a phospholipase C and elevates [Ca2+]i. Furthermore, we present evidence that hydrolysis of PtdIns(4,5)P2 is not secondary to the elevation of [Ca2+]i. TRH caused a rapid decrease in the level of PtdIns(4,5)P2 to 57% of control and stimulated an increase in inositoltriphosphate, the unique product of phospholipase C-mediated hydrolysis of PtdIns(4,5)P2, to a peak of 280% of control. In control cells, resting [Ca2+]i was 106 +/- (SE) 27 nM, and TRH stimulated a rapid elevation to 700 +/- 210 nM. In experiments performed to determine whether PtdIns(4,5)P2 hydrolysis induced by TRH may have been caused by the elevation of [Ca2+]i, the following results were obtained: the effect of TRH to decrease the level of PtdIns(4,5)P2 was not reproduced by the calcium ionophore A23187 or by membrane depolarization with 50 mM K+; the calcium antagonist TMB-8 did not inhibit the TRH-induced decrease in PtdIns(4,5)P2; and, most importantly, inhibition by EGTA of the elevation of [Ca2+]i did not inhibit the TRH-induced decrease in PtdIns(4,5)P2. We suggest that phospholipase C-mediated hydrolysis of PtdIns(4,5)P2 to yield inositoltriphosphate may be the initial event in TRH action in TtT cells, as in GH3 cells, that leads to elevation of [Ca2+]i and to TSH secretion.

Poly(A)-containing RNA isolated from bovine and mouse pituitaries and a mouse pituitary thyrotropic tumor was translated in a wheat germ cell-free biosynthetic system. A precursor of the glycoprotein hormone alpha subunit, "pre-alpha," was immunoprecipitated from the translation mixtures with antiserum against bovine luteinizing hormone (LH; lutropin) alpha. The specificity of the immunoprecipitation was shown by competition with authentic bovine LHalpha and lack of competition with bovine thyroid-stimulating hormone (TSH; thyrotropin) beta. Bovine and mouse pre-alpha subunits migrated identically in sodium dodecyl sulfate gradient polyacrylamide slab gels with an apparent molecular weight of about 17,000. Pre-alpha comprised 2-3% and 20-30% of the total proteins translated with pituitary and pituitary tumor mRNA, respectively. Microanalysis of amino acid sequence of the pre-alpha subunits containing various radiolabeled amino acids gave the following partial sequence for mouse tumor pre-alpha: [Formula: see text] Met was also found in positions 1, 14, and 17 in mouse pituitary pre-alpha but only in residue 1 of the bovine pituitary pre-alpha subunit. Leu was found in identical positions in bovine pituitary pre-alpha, with an additional Leu in position 17. Leu in the common positions (12, 15, 19, and 22) has also been found in human choriogonadotropin pre-alpha subunit [Birken, S., Fetherston, J., Desmond, J., Canfield, R. & Boime, I. (1978) Biochem. Biophys. Res. Commun. 85, 1247-1253]. The data demonstrate that pituitary as well as placental glycoprotein hormone alpha subunits are synthesized with an amino-terminal hydrophobic extension, in accord with the "signal hypothesis" for secreted proteins. Furthermore, the positions of the hydrophobic amino acid Leu have been strictly conserved in pre-alpha subunits from various species and in two different tissues, the pituitary and placenta.

Studies of TSH release and production were performed in short term monolayer cultures of transplantable, thyroid hormone responsive, thyrotropin (TSH) producing mouse pituitary tumors. These tumors contained large amounts of TSH, small amounts of growth hormone (GH) and no detectable luteinizing hormone (LH), indicating that the predominant hormone product of tumor cells was TSH. The TSH content per tumor cell was similar to that of the normal pituitary where thyrotrophs represent a small fraction of the total cells, suggesting that the TSH content per tumor cell was less than that of the normal thyrotroph. There was a time dependent release and production of TSH by tumor cells in monolayer culture. Thyrotropin releasing hormone (TRH) increased the release into the media and the production of TSH in a dose dependent manner. Maximum effects were noted at 0.2 ng/ml. Thyroid hormones and somatostatin inhibited both basal and TRH induced effects on both TSH release and production. TSH release as induced by TRH was calcium dependent. TSH release was stimulated by ouabain (10(-3)M) and potassium (57 mM), agents known to promote cellular calcium uptake in a calcium dependent manner. These studies indicate that tumor derived cells function in monolayer culture in a similar fashion to normal thyrotrophs. Studies were conducted to test the hypothesis that TRH action is mediated by adenosine 3',5' monophosphate (cAMP). Dibutyryl cAMP (6 mM) and theophylline (10 mM) increased TSH release suggesting that cAMP is involved in TSH release. However, TRH had no detectable effect on tumor cell adenylate cyclase activity or levels of cAMP. In contrast, PGE1 (1-10 mug/ml) stimulated adenylate cyclase activity and elevated cellular levels of cAMP without increasing TSH release. Thus, we are unable to confirm the postulate that cAMP is the intracellular mediator of TRH action.