Efatutazone, an oral PPAR-γ agonist, in combination with paclitaxel in anaplastic thyroid cancer: results of a multicenter phase 1 trial.

PURPOSE: A phase 1 study was initiated to determine the safety, potential effectiveness, and maximal tolerated dose and recommended phase 2 dose of efatutazone and paclitaxel in anaplastic thyroid cancer. EXPERIMENTAL DESIGN: Patients received efatutazone (0.15, 0.3, or 0.5 mg) orally twice daily and then paclitaxel every 3 weeks. Patient tolerance and outcomes were assessed, as were serum efatutazone pharmacokinetics. RESULTS: Ten of 15 patients were women. Median age was 59 years. Seven patients received 0.15 mg of efatutazone, 6 patients received 0.3 mg, and 2 patients received 0.5 mg. One patient receiving 0.3 mg of efatutazone had a partial response from day 69 to day 175; 7 patients attained stable disease. Median times to progression were 48 and 68 days in patients receiving 0.15 mg of efatutazone and 0.3 mg of efatutazone, respectively; corresponding median survival was 98 vs 138 days. The median peak efatutazone blood level was 8.6 ng/mL for 0.15-mg dosing vs 22.0 ng/mL for 0.3-mg twice daily dosing. Ten patients had grade 3 or greater adverse events (Common Terminology Criteria for Adverse Events), with 2 of these (anemia and edema) related to efatutazone. Thirteen events of edema were reported in 8 patients, with 2 of grade 3 or greater. Eight patients had ≥1 serious adverse event, with 1 of these (anemia) attributed to efatutazone and 1 (anaphylactic reaction) related to paclitaxel. The maximal tolerated dose was not achieved. Angiopoietin-like 4 was induced by efatutazone in tissue biopsy samples of 2 patients. CONCLUSIONS: Efatutazone and paclitaxel in combination were safe and tolerated and had biologic activity.

Undetectable sensitive serum thyroglobulin (<0.1 ng/ml) in 163 patients with follicular cell-derived thyroid cancer: results of rhTSH stimulation and neck ultrasonography and long-term biochemical and clinical follow-up.

CONTEXT: Surveillance of patients with differentiated thyroid cancer (DTC) is achieved using serum thyroglobulin (Tg), neck ultrasonography (US), and recombinant human TSH (rhTSH)-stimulated Tg (Tg-stim). OBJECTIVE: Our primary aim was to assess the utility of rhTSH Tg-stim in patients with suppressed Tg (Tg-supp) below 0.1 ng/ml using a sensitive assay. Our secondary aims were to assess the utility of US and to summarize the profile of subsequent Tg-supp measures. DESIGN: This is a retrospective study conducted at two sites of an academic institution. PATIENTS: A total of 163 patients status after thyroidectomy and radioactive iodine treatment who had Tg-supp below 0.1 ng/ml and rhTSH Tg-stim within 60 d of each other were included. RESULTS: After rhTSH stimulation, Tg remained below 0.1 ng/ml in 94 (58%) and increased to 0.1-0.5 in 56 (34%), more than 0.5-2.0 in nine (6%), and above 2.0 ng/ml in four (2%) patients. Serial Tg-supp levels were obtained in 138 patients followed over a median of 3.6 yr. Neck US were performed on 153 patients; suspicious exams had fine-needle aspiration (FNA). All positive FNA were identified around the time of the initial rhTSH test. Six of seven recurrences were detected by US (Tg-stim >2.0 ng/ml in one, 0.8 in one and ≤ 0.5 in four). One stage IV patient had undetectable Tg-stim. CONCLUSION: In patients with DTC whose T(4)-suppressed serum Tg is below 0.1 ng/ml, long-term monitoring with annual Tg-supp and periodic neck US are adequate to detect recurrences. In our experience, rhTSH testing does not change management and is not needed in this group of patients.

Anaplastic thyroid carcinoma: pathogenesis and emerging therapies.

Anaplastic thyroid carcinoma ranges from 1.3 to 9.8% of all thyroid cancers globally. Mutations, amplifications, activation of oncogenes and silencing of tumour suppressor genes contribute to its aggressive behaviour, and recent studies (e.g. microarrays, microRNAs) have provided further insights into its complex molecular dysregulation. Preclinical studies have identified numerous proteins over- or underexpressed that affect critical cellular processes, including transcription, signalling, mitosis, proliferation, cell cycle, apoptosis and adhesion, and a variety of agents that effectively inhibit these processes and tumour growth. In clinical studies of 1771 patients, 64% were women, the median survival was 5 months, and 1-year survival was 20%. The variables associated with survival in some series included age, tumour size, extent of surgery, higher dose radiotherapy, absence of distant metastases at presentation, co-existence of differentiated thyroid cancer and multimodality therapy. However, considerable bias exists in these non-randomised studies. Although more aggressive radiotherapy has reduced locoregional recurrences, the median overall survival has not improved in over 50 years. Newer systemic therapies are being tried, and more effective combinations are needed to improve patient outcomes.

Hormonal and cardiovascular response to low-intensity exercise with atropine administration.

The hormonal and cardiovascular responses to atropine and low-intensity exercise were examined in 7 young men. Subjects completed 3 trials in a single blind crossover design. During the first trial (T1), subjects received 2.0 mg of atropine intramuscularly at rest. Subsequently in trial 2 (T2), subjects received a saline placebo before 90 minutes of intermittent exercise, and during trial 3 (T3), they received atropine before 90 min of intermittent exercise [3 x (25-minute cycle/5-minute rest) @ 40% VO2 peak]. Venous blood samples and physiological data were collected before, during, and post exercise. Growth hormone (GH) was significantly increased in T2 but unchanged in T1 and T3. Cortisol (CORT) was unchanged in T1 and T2, but in T3 significantly increased (p <0.05) from 45 to 90 minutes compared to T2. Plasma luteinizing hormone (LH) was unaffected in all trials. Plasma prolactin (PRO) significantly increased in T3 from 45 to 90 minutes in comparison to T2. Norepinephrine (NE) was unaffected in T1, but significantly increased in both T2 and T3 (5 to 90 minutes). NE in T3 was also significantly higher compared to T2 (30 to 90 minutes). The heart rate (HR) and rate pressure product (RPP) significantly increased in all trials (15 to 90 minutes) and T3 was significantly elevated in comparison to T2. The administration of atropine before 90 minutes of low-intensity exercise significantly increased cortisol, prolactin, and norepinephrine, decreased growth hormone, and significantly increased cardiovascular stress.

Cinacalcet hydrochloride reduces the serum calcium concentration in inoperable parathyroid carcinoma.

BACKGROUND: Management of inoperable parathyroid carcinoma presents a challenge because until recently, effective medical therapy was not available. Morbidity and mortality result primarily from severe hypercalcemia. We assessed the ability of the calcimimetic cinacalcet HCl to reduce serum calcium in patients with parathyroid carcinoma as well as its effect on PTH concentrations, bone turnover markers, safety, and health-related quality of life variables. METHODS: Twenty-nine patients with parathyroid carcinoma were enrolled in this open-label, single-arm study consisting of titration and maintenance phases. Cinacalcet doses were titrated (30 mg twice daily to 90 mg four times daily) for 16 wk or until serum calcium was no more than 10.0 mg/dl. The study endpoint was the proportion of patients with at least a 1 mg/dl reduction in serum calcium at the end of the titration phase (responders). RESULTS: Mean (+/- se) serum calcium (14.1 +/- 0.4 mg/dl) and PTH (697 +/- 94 pg/ml) were markedly elevated at baseline. At the end of the titration period, serum calcium was reduced by at least 1 mg/dl in 62% of patients (mean decline to 12.4 +/- 0.5 mg/dl). In the 18 responders, serum calcium fell from 15.0 +/- 0.5 to 11.2 +/- 0.3 mg/dl (P < 0.001). The greatest reductions in serum calcium were observed in patients with highest baseline calcium levels. PTH levels decreased, but not significantly, to 635 +/- 73 pg/ml (-4.6%). Adverse events included nausea, vomiting, headache, and fracture. CONCLUSIONS: Cinacalcet effectively reduces hypercalcemia in approximately two thirds of patients with inoperable parathyroid carcinoma and may represent an important new treatment option for these patients.

Novel high-affinity PPARgamma agonist alone and in combination with paclitaxel inhibits human anaplastic thyroid carcinoma tumor growth via p21WAF1/CIP1.

Peroxisome proliferator-activated receptor gamma (PPARgamma) agonists demonstrate antitumor activity likely through transactivating genes that regulate cell proliferation, apoptosis, and differentiation. The PAX8/PPARgamma fusion oncogene, which is common in human follicular thyroid carcinomas appears to act via dominant negative suppression of wild-type PPARgamma, suggesting that it may be a tumor suppressor gene in thyroid cells. We have identified a novel high-affinity PPARgamma agonist (RS5444) that is dependent upon PPARgamma for its biological activity. This is the first report of this molecule and its antitumor activity. In vitro, the IC50 for growth inhibition is approximately 0.8 nM while anaplastic thyroid carcinoma (ATC) tumor growth was inhibited three- to fourfold in nude mice. siRNA against PPARgamma and a pharmacological antagonist demonstrated that functional PPARgamma was required for growth inhibitory activity of RS5444. RS5444 upregulated the cell cycle kinase inhibitor, p21WAF1/CIP1. Silencing p21WAF1/CIP1 rendered cells insensitive to RS5444. RS5444 plus paclitaxel demonstrated additive antiproliferative activity in cell culture and minimal ATC tumor growth in vivo. RS5444 did not induce apoptosis but combined with paclitaxel, doubled the apoptotic index compared to that of paclitaxel. Our data indicate that functional PPARgamma is a molecular target for therapy in ATC. We demonstrated that RS5444, a thiazolidinedione (Tzd) derivative, alone or in combination with paclitaxel, may provide therapeutic benefit to patients diagnosed with ATC.

Cushing's syndrome due to medullary thyroid carcinoma: diagnosis by proopiomelanocortin messenger ribonucleic acid in situ hybridization.

Medullary thyroid carcinoma (MTC) rarely causes ectopic ACTH syndrome. We describe a 38-yr-old man with renal stones who had a 5-cm MTC removed in 1992. He was RET-protooncogene positive (codon 618). Serum calcitonin was 1597 pg/ml postoperatively. In 1996 he had rib fractures, bruising, weakness, and three to four stools per day. Laboratory studies revealed an elevated 24-h urine-free cortisol (780 micro g/d), epinephrine (66 micro g/d), and calcium (558 mg/d). Baseline serum cortisol was 23.9 micro g/dl and decreased to 12.9 and 4.5 micro g/dl after 2 mg and 8 mg dexamethasone suppression, respectively. Plasma ACTH was 170 pg/ml and decreased to 75 and 24 pg/ml after dexamethasone. Bone density t-score was -4.3 (trochanter). Computed tomography scans showed multiple cervical nodes and 2-cm right adrenal nodule. Magnetic resonance imaging (MRI) scan showed a prominent, homogeneous pituitary; the adrenal MRI scan was not typical for a pheochromocytoma. Serum CRH was less than 6.6 pg/ml. Bilateral adrenalectomy revealed two adjacent right adrenal pheochromocytomas and corrected the elevated urine cortisol (30 micro g/d), epinephrine (0 micro g/d), and calcium (281 mg/d) but not plasma ACTH (125 pg/ml). Neck dissection reduced calcitonin by 96% (5300 to 120 pg/ml) and ACTH by 91% (125 to 11 pg/ml). Carcinoembryonic antigen was reduced from 32.0 to 2.3 ng/ml. Immunohistochemical stain was negative for ACTH in the MTC-positive lymph nodes and the pheochromocytoma. Proopiomelanocortin mRNA by in situ hybridization was positive in the MTC but not in the pheochromocytoma. A repeat pituitary MRI scan was normal. The differential diagnosis of ACTH-dependent Cushing's syndrome in this case included pituitary disease or ectopic ACTH, either from medullary thyroid carcinoma or pheochromocytoma. ACTH stains were unrevealing, but proopiomelanocortin mRNA in situ hybridization in MTC tissue and plasma ACTH response to neck dissection confirmed MTC as the source of ectopic ACTH.

Renal metastases from thyroid papillary carcinoma: study of sodium iodide symporter expression.

Kidney metastases from thyroid cancer are rare. We report two such patients and demonstrate that the in vivo 131I uptake by the kidney metastasis is associated with high levels of sodium iodide (Na+/I-) symporter (NIS) expression in the first case. Case 1: A 61-year-old woman with papillary thyroid carcinoma-follicular variant (PTC-FV) presented with scapular metastasis. After thyroidectomy and scapulectomy, a 131I posttherapy scan showed left upper quadrant uptake. A 3.0-cm metastatic PTC-FV deposit was removed by partial nephrectomy. Case 2: A 53-year-old woman presented with back pain. A computed tomography (CT) scan showed a 3.5-cm renal mass, a multinodular goiter, and lung metastases thought secondary to a renal cell carcinoma. A unilateral nephrectomy revealed metastatic PTC-FV. After thyroidectomy, a 131I posttherapy scan showed lung and skeletal metastases. NIS immunoreactivity in tumoral tissue was strongly positive in the primary tumor, shoulder, and kidney metastasis in case 1, as well as in the primary tumor in case 2. Spotty, low-level NIS expression was observed in the kidney metastasis in case 2. In conclusion, kidney metastases of PTC-FV may occasionally retain adequate levels of NIS expression, enabling their detection during life. Thus, intense uptake in the abdomen during 131I imaging should not be assumed to be physiological gastrointestinal tract residual radionuclide activity.

Human telomerase reverse transcriptase expression in Diff-Quik-stained FNA samples from thyroid nodules.

Fine-needle aspiration (FNA) is a highly sensitive method in the differential diagnosis of thyroid nodules. However, 10% of thyroid FNAs are indeterminate for cancer, and thus additional markers may be useful diagnostically. The authors have demonstrated previously that human telomerase reverse transcriptase (hTERT) gene expression is useful in the distinction of benign lesions from malignant lesions. They therefore wondered whether the detection of hTERT gene expression was feasible using archival slides. To establish an experimental system, ribonucleic acid was extracted from human anaplastic thyroid carcinoma cell line (ARO) in cytologic specimens, and reverse transcription-polymerase chain reaction (RT-PCR) for hTERT expression was performed. RT-PCR analysis for hTERT gene detection was then performed using 58 Diff-Quik-stained archival FNA samples collected retrospectively. RT-PCR for human thyroglobulin (hTg) or beta-actin gene expression served as a positive control. Successful PCR results were obtained from 48 of the 58 cases. All 10 slides in which no RT-PCR products were noted were older than 3 years. hTERT gene expression was demonstrated in FNAs from two of seven cases (29%) of hyperplastic nodule, one of one case (100%) of Hashimoto's thyroiditis, three of eight cases (38%) of follicular adenoma, three of eight cases (38%) of Hürthle cell adenoma, three of four cases (75%) of follicular carcinoma, two of two cases (100%) of Hürthle cell carcinoma, and 11 of 18 cases (61%) of papillary carcinoma. All but one of the available 33 corresponding frozen samples exhibited the same RT-PCR results. This study demonstrates that Diff-Quik-stained thyroid FNA specimens less than 3 years old can be used for the detection of hTERT gene expression by RT-PCR. This test, along with careful cytopathologic examination, may improve our ability to differentiate benign lesions from malignant lesions in indeterminate FNA samples from thyroid nodules.

Simultaneous thalamic deep brain stimulation and implantable cardioverter-defibrillator.

Thalamic deep brain stimulation is becoming increasingly popular for the control of drug-refractory tremor. Implantable cardiac pacemakers and defibrillators are commonly used therapeutic modalities. Concerns exist about the potential interactions between these 2 devices in the same patient, but no experience has been reported previously. We describe a patient with essential tremor who had a deep brain stimulator implanted into the left ventral intermediate nucleus of thalamus, who subsequently needed an implantable cardioverter-defibrillator. Despite concerns about possible interactions between the 2 types of implanted electrical devices (i.e., a situation similar to drug-drug interactions), the deep brain stimulator and the implanted pacemaker-defibrillator functioned appropriately, and no interaction occurred in our patient.

Treatment of a thyrotropinoma with octreotide-LAR in a patient with multiple endocrine neoplasia-1.

OBJECTIVE: To note that a thyrotropin (TSH)-secreting macroadenoma may be part of the multiple endocrine neoplasia-1 (MEN-1) syndrome and to report the use of octreotide-LAR (OCT-LAR) to treat a TSH-secreting macroadenoma in a patient with MEN-1 with previous surgery for hyperparathyroidism and gastrinoma. METHODS: We present a patient with a TSH-secreting pituitary macroadenoma and report the results of her endocrine, genetic, radiologic, and nuclear medicine testing and her response to treatment with octreotide (OCT), octreotide-LAR, and estrogen. RESULTS: This patient's TSH-induced hyperthyroidism responded to octreotide for 5 months and octreotide-LAR for more than 11 months. Her hypercalcemia normalized while she was taking estrogen. Her genetic testing is reported to show a genetic defect that is typical of patients with MEN-1. CONCLUSION: This report describes: (1) The use of octreotide-LAR to treat both a TSH-secreting pituitary tumor and a gastrinoma over 12 months; (2) the importance of including these tumors into the MEN-1 syndrome with its attendant implications; and (3) a genetic defect, typical of patients with MEN-1, associated with this tumor.

Corticotropin- and thyrotropin-secreting pituitary microadenomas: detection by dynamic magnetic resonance imaging.

Hormone-secreting pituitary microadenomas are often not visible on magnetic resonance imaging (MRI). Diagnosis requires confirmatory endocrine test results and often an invasive procedure, inferior petrosal sinus sampling (IPSS). Improved pituitary imaging may eliminate the need for IPSS in some patients, as shown in the 2 women in this report. The first patient with hirsutism, weight gain, and hypertension had intermittent elevations of urinary free cortisol, abnormal results on a low-dose dexamethasone suppression test, and positive results on a dexamethasone-suppressed ovine corticotropin-releasing hormone test (corticotropin, increase of 122%; cortisol, increase of 118%). Gadolinium-enhanced MRI showed no focal lesion, but dynamic MRI (sequential images beginning immediately after contrast injection) revealed a right-sided 5-mm microadenoma, confirmed by transsphenoidal surgery. The second patient had a goiter, anxiety, increased free thyroxine and triiodothyronine levels, and a normal thyrotropin value with no response to thyrotropin-releasing hormone. Magnetic resonance imaging showed no lesion, but dynamic MRI detected an 8-mm microadenoma. Although about 8% to 10% of healthy persons have incidental pituitary lesions that are 3 mm or larger on MRI, identification of a distinct lesion and positive results on a dexamethasone-suppressed ovine corticotropin-releasing hormone test should decrease the probability of a false-positive result on an imaging study. We recommend that dynamic MRI be performed in any patient with a suspected microadenoma, before IPSS is performed.

Disclosing subclinical thyroid disease. An approach to mild laboratory abnormalities and vague or absent symptoms.

Patients with subclinical thyroid disease often have no apparent symptoms or only nonspecific complaints. However, increasing evidence that early disease is associated with behavioral, psychiatric, biochemical, and organ-specific abnormalities has led several specialty organizations to publish or modify position papers. Serum TSH testing is the most sensitive method of identifying early thyroid dysfunction. It should be considered in patients who have risk factors for mild thyroid failure, have symptoms that could be related to thyroid disease, or are taking exogenous thyroid hormone. T4 therapy should be strongly considered in patients with a TSH level of 10 mIU/L or more. If observation is elected in asymptomatic patients with lesser TSH elevation, periodic measurements are advised. In patients with TSH suppression who are taking thyroid hormone, the dose should be lowered. If the TSH level is decreased because of endogenous suppression and free-T4 and T3 levels are normal, options include observation and treatment with an antithyroid drug or thyroid ablation. Early therapy should be considered in older patients and those with heart disease or nodular thyroid disease. The goal of all treatment methods should be to keep the TSH level in the normal range.

Human telomerase reverse transcriptase (hTERT) gene expression in FNA samples from thyroid neoplasms.

BACKGROUND: Although fine-needle aspiration (FNA) is the most sensitive method for the detection of thyroid carcinoma, it cannot provide a definitive diagnosis of malignancy in 60% of the patients operated on for suspicious lesions. Recently, human telomerase reverse transcriptase (hTERT) has been found to be a diagnostic marker of malignancy. We therefore sought to determine whether hTERT gene expression could serve as an adjunct to FNA in the differential diagnosis of thyroid nodules. METHODS: Twenty-four FNA samples from thyroid nodules that were suspected of malignancy were collected. RNA was extracted, and hTERT gene expression was examined by RT-PCR. Cytologic and histologic examinations were also performed. RESULTS: Two of three follicular, three of three Hürthle cell, and eight of eight papillary thyroid carcinomas had corresponding FNA samples that were positive for hTERT. One of two Hürthle cell adenomas was hTERT positive. FNA samples from three follicular adenomas and five hyperplastic nodules were negative for hTERT. Positive and negative predictive values were 93% and 90%, respectively. CONCLUSIONS: The detection of hTERT gene expression in thyroid FNA samples holds promise as a diagnostic marker in the distinction of benign from malignant thyroid lesions. Its application could alter the surgical management of these patients.

Characterization of distinct heat shock- and thapsigargin-induced cytoprotective proteins in FRTL-5 thyroid cells.

Heat shock induces the expression of proteins with molecular weights of 70-72 kd and 90 kd, whereas thapsigargin induces the expression of a glucose-regulated protein 78 kd (GRP-78) in certain cells. In this study we examined the induction and cytoprotective effects of heat shock- and thapsigargin-induced proteins in FRTL-5 rat thyroid cells. New protein synthesis was assessed in [35S]methionine-labeled cells and quantitated densitometrically. The expression of specific stress proteins was identified using Western blots, whereas cytoprotection provided by these proteins was evaluated by trypan blue exclusion. Exposure to heat shock (45 degrees C, 15 minutes) induced the expression of proteins with molecular weights at the range of low 70 kD and low 90 kD that peaked between 2-6 hours and returned to baseline within 24 hours. Treatment of cells with thapsigargin (200 nM, 15 minutes) induced the expression of different molecular weight proteins, most likely GRP-78 and -94, that peaked at 4-6 hours and lasted for 24 hours. Neither the removal of growth factors (thyroid-stimulating hormone and insulin) for 5 days nor the elimination of extracellular Ca2+ with EGTA or clamping of the intracellular Ca2+ with BAPTA for 15 minutes affected expression of the heat shock- and the thapsigargin-induced stress proteins. In contrast, protein kinase C inhibitors H7 and GF109203X abolished the expression of all three groups of stress proteins. Both heat shock- and thapsigargin-inuced proteins completely protected cells from subsequent thermal injury (47 degrees C, 35 minutes). The induction of cytoprotective proteins by heat shock and thapsigargin requires the presence of protein kinase C but is Ca(2+)- and growth factor-independent.

Phospholipase C activation by Na+/Ca2+ exchange is essential for monensin-induced Ca2+ influx and arachidonic acid release in FRTL-5 thyroid cells.

BACKGROUND: Monensin, a Na+ ionophore, can increase cytosolic Ca2+ ([Ca2+]i) by reversing the Na+/Ca2+ exchange mechanism. This study provided additional insights into the mechanism of this Na+ ionophore-induced increase in [Ca2+]i, and emphasized the critical role of phospholipase C (PLC) in amplifying Na+/Ca2+ exchange-induced Ca2+ influx and subsequent arachidonic acid (AA) release in FRTL-5 thyroid cells. The possible involvement of protein kinase C (PKC), mitogen-activated protein kinase (MAPK), and GTP-binding (G) protein in mediating monensin-induced AA release was also explored. METHODS: FRTL-5 thyroid cells were maintained in Coon's modified Ham's F-12 medium supplemented with a 6-hormone (6H) mixture. Cytosolic Ca2+ was measured by using indo-1 AM and a dual-wave-length spectrofluorometer. Release of 3H-labeled inositol trisphosphates and arachidonic acid were determined by a scintillation counter. RESULTS: In Hank's balanced salt solution with Ca2+ (HBSS+), monensin (100 mumol/L) induced a 2.3-fold sustained Ca2+ increase associated with IP3 generation and a 6-fold increase in AA release. Deletion of extracellular Ca2+, or replacement of Na+ by choline chloride in the medium, reduced the [Ca2+]i increase by 77% and completely prevented the monensin-induced rise in AA release. Similar inhibitory effects were observed in cells pretreated with a Na+ channel blocker, or Na+/Ca2+ exchange inhibitors. In HBSS without Ca2+ (HBSS-), monensin induced a 1-fold transient [Ca2+]i increase but did not increase the AA. This Ca2+ increase was not suppressed by U-73122, a PLC inhibitor. In HBSS+, U-73122 did not affect the monensin-induced initial transient peak increase of [Ca2+]i, but reduced the sustained second phase of [Ca2+]i from 400 nmol/L to 250 nmol/L, and completely blocked AA release. A phospholipase A2 (PLA2) inhibitor blocked the monensin-induced AA release without affecting the [Ca2+]i increase. Inhibition of PKC prevented 87% to 94% of the monesin-stimulated AA release. The monensin-induced AA release was also inhibited 94% by pertussis and 51% by a MAP kinase cascade inhibitor. CONCLUSIONS: The results suggest that monensin initiates an increase in [Ca2+]i via a Na+/Ca2+ exchange mechanism that triggers more pronounced and sustained [Ca2+]i increase via activation of PLC and Ca2+ influx. The PLC activation, followed by sustained Ca2+ influx and PKC activation, is a prerequisite for PLA2-mediated processes in monensin-challenged FRTL-5 thyroid cells.