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Institut National de la Santé et de la Recherche Médicale (INSERM) U413 (V.C., E.L., C.D., D.C., F.S., J.-M.K., H.V., H.L.), Laboratory of Cellular and Molecular Neuroendocrinology, European Institute for Peptide Research (Institut Fédératif de Recherches Multidisciplinaires sur les Peptides 23), University of Rouen, 76821 Mont-Saint-Aignan, France; Department of Endocrinology (Y.R.), Centre Hospitalier Universitaire of Caen, 14033 Caen, France; Center for Clinical Investigation (C.D., J.-M.K., H.L.), INSERM Centre d’Investigation Clinique 204 and Department of Pathology (A.L.), University Hospital of Rouen, 76031 Rouen, France; and Laboratory of Oncologic Genetics (F.P.), Centre Henri Becquerel, 76038 Rouen, France
Address all correspondence and requests for reprints to: Dr. Hervé Lefebvre, Institut National de la Santé et de la Recherche Médicale Unité 413, Institut Fédératif de Recherche Multidisciplinaires sur les Peptides 23, Department of Endocrinology, Hospital of Boisguillaume, Centre Hospitalier Universitaire of Rouen, 76031 Rouen cedex, France. E-mail: herve.lefebvre{at}chu-rouen.fr.
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Abstract |
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Several observations indicate that cortisol production is controlled by illegitimate (or aberrant) membrane receptors in both adrenal adenomas and ACTH-independent macronodular adrenal hyperplasias (AIMAHs) causing Cushing’s syndrome (8). These aberrant receptors include ectopic receptors for gastric inhibitory polypeptide, LH, or catecholamines and overexpressed eutopic receptors like vasopressin V1a and serotonergic 5-HT4 receptors (8). In particular, an abnormal cortisol response to administration of metoclopramide and/or cisapride due to adrenocortical overexpression of the 5-HT4 receptor has been observed in patients with AIMAH (9). Similarly, cisapride has been recently found to aberrantly stimulate cortisol secretion in some patients with adrenocortical adenomas causing subclinical or overt Cushing’s syndrome (10, 11). However, the sensitivity of the adrenocortical tissue to 5-HT and/or serotonergic agonists has never been investigated in vitro in cortisol-producing adrenal tumors.
In the present study, we report two cases of cortisol-secreting adenomas abnormally responsive in vivo to cisapride administration. After surgery, in vitro experiments were conducted to investigate the effect of 5-HT on both perifused tumor explants and cultured tumor cells. In vivo and in vitro studies also revealed that the two tumors simultaneously expressed several illegitimate receptors. Notably, we describe the first observation of a cortisol-producing adrenocortical lesion abnormally sensitive to glucagon and GnRH.
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Patients and Methods |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Two patients with incidentally discovered unilateral adrenal adenoma were studied.
Patient 1, a 72-yr-old woman was referred for a tumor of the left adrenal gland discovered during a computed tomography (CT) scan performed for abdominal pain. She had no history of hypertension. Clinical examination revealed no sign of hypercortisolism. On abdominal CT scan, the adrenal mass size was 3.2 cm with an attenuation value of 10 HU. An initial ambulatory endocrine evaluation revealed that urinary free cortisol (UFC), metanephrine, and normetanephrine were normal, i.e. reaching, respectively, 23 µg/d [63 nmol/d, normal value (N) < 80 µg/d (220 nmol/d)], 80 µg/d [405 nmol/d, N < 300 µg/d (1520 nmol/d)], and 102 µg/d [1500 nmol/d, N < 400 µg/d (2180 nmol/d)]. The tumor was considered as a nonfunctioning adenoma, and the patient was reevaluated 6 months later in our Department of Endocrinology. A 0.4-cm increase in mass diameter was then observed on CT scan, and hormonal investigations gave the following results: plasma cortisol levels were 13.3 µg/dl [367 nmol/liter, N = 8–25 µg/dl (220–690 nmol/liter)] at 0800 h, 4.3 µg/dl (118 nmol/liter) at 1200 h, and 3.0 µg/dl (82 nmol/liter) after an overnight 1-mg dexamethasone suppression test. Plasma ACTH level was 8 pg/ml [1.72 pmol/liter, N = 10–80 pg/ml (2.2–17.5 pmol/liter)] at 0800 h, and UFC was 23 µg/d [63 nmol/d, N < 80 µg/d (220 nmol/d)]. Mass enlargement as well as the biological data suggestive of subclinical Cushing’s syndrome led to surgical removal of the tumor. Histopathological examination of the lesion confirmed the diagnosis of adrenocortical adenoma.
Patient 2, a 48-yr-old man, was referred for a tumor of the right adrenal gland discovered during CT scan performed for lumbar pain. He had no history of hypertension or diabetes. Clinical examination revealed no sign of hypercortisolism. On abdominal CT scan, the tumor presented as a heterogenous 3.7-cm mass containing hypodense and hyperdense areas with attenuation values of 5 HU and 39 HU, respectively. Endocrine investigations showed the following results: plasma cortisol levels were 18.0 µg/dl [495 nmol/liter, N = 8–25 µg/dl (220–690 nmol/liter)] at 0800 h, 4.5 µg/dl (124 nmol/liter) at 1200 h, and 1.5 µg/dl (41 nmol/liter) after an overnight 1-mg dexamethasone suppression test. Plasma ACTH level was 19 pg/ml [4.20 pmol/liter, N = 10–80 pg/ml (2.2–17.5 pmol/liter)] at 0800 h, and UFC was 82 µg/d [226 nmol/d, N < 100 µg/d (275 nmol/d)]. An iodocholesterol scan performed without dexamethasone suppression showed a unilateral uptake of the tracer by the tumor suggestive of autonomous cortisol production. Dual-energy x-ray absorptiometry revealed a significant reduction in bone mineral density (t-score –2.59 at lumbar spine). The patient underwent right adrenalectomy, and the diagnosis of adrenocortical adenoma was confirmed by pathological examination.
Clinical investigation protocol
Abnormal sensitivity of the tumor tissue to physiological and pharmacological stimuli was systematically searched for by using a clinical protocol performed on 3 consecutive days after the initial routine hormonal investigations, as previously described (11). The protocol was approved by the regional ethics committee, and the two patients gave their informed consent. Briefly, the patients were given 2 mg dexamethasone orally at 0800, 1400, 2000, and 2400 h from d 1–3 to investigate the cortisol response to various stimuli independently of any ACTH variation. On d 2, plasma cortisol levels were measured in response to a posture test; a mixed 1030-kcal standard meal containing 50% carbohydrates, 26% protein, and 24% fat; and a combined TRH (200 µg)/GHRH (80 µg)/GnRH (100 µg) iv test, all three hypothalamic hormones being purchased from Ferring (Gentilly, France). Then the cortisol response to iv administration of 1 mg of the lysine vasopressin precursor terlipressin (Glypressine; Ferring) was investigated in patient 2 but not in patient 1 whose age, i.e. above 70 yr, contraindicated this test. On d 3, the patients underwent glucagon (1 mg iv, Glucagen; Novo-Nordisk, Boulogne-Billancourt, France), cisapride (10 mg orally, Prepulsid; Janssen-Cilag, Issy-les Moulineaux, France), and cosyntropin (0.25 mg iv, Synacthène; Novartis, Rueil Malmaison, France) stimulation tests. Plasma cortisol levels were measured by RIA (DiaSorin CA 1529–49, Antony, France), and plasma ACTH levels were measured by immunoradiometric assay (Nichols Institute, San Juan Capistrano, CA). All results of the tests are expressed as percent variations from basal, calculated as follows: peak cortisol-basal cortisol/basal cortisol x 100. Cortisol responses to stimulation were arbitrarily classified as negative, weak, moderate, or strong if the increase in plasma cortisol from baseline was less than 25, 25–49, 50–99, or 100% or more, respectively.
In vitro studies
Tissue collection. Tumor tissues were obtained at surgery and immediately dissected by the pathologist. Normal adrenal explants (control tissues) were obtained from patients undergoing expanded nephrectomy for kidney cancer. Adrenocortical fragments were transported to the laboratory in culture medium for primary culture and perifusion experiments, frozen on dry ice and stored at –80 C until real-time RT-PCR experiments, or fixed in formalin and embedded in paraffin for immunohistochemical analysis (see below). The protocol of collection of the tissues and the experimental procedures were approved by the regional ethics committees, and written informed consent was obtained from the patients.
Reagents. Protease, collagenase (type IA), deoxyribonuclease I, insulin, apo-transferrin, L-ascorbic acid, ACTH, 5-HT, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H89), methiothepine, and the rabbit 5-HT antiserum were purchased from Sigma (St-Quentin-Fallavier, France). The nutrient medium F-12 (Ham) and DMEM were obtained from Life Technologies, Inc. (Paisley, Scotland, UK). The antibiotic-antimycotic solution and fetal bovine serum were from Bio-Whittaker (Walkersville, MD). GnRH, TRH, and GHRH were purchased from Ferring. Glucagon was from Novo-Nordisk, and human chorionic gonadotropin (hCG) was from Organon (Eragny-sur-Epte, France). GR 113808 was from GlaxoSmithKline (Greenford, England, UK).
Cell culture. Adrenocortical adenoma and normal adrenal gland fragments were immersed in culture medium (50% DMEM-50% Ham’s F-12) supplemented with 0.2% antibiotic-antimycotic solution and rapidly transported to the laboratory. The adrenal cortex was dissected from fat and medullary tissues, minced with scissors, and adrenal cells enzymatically dispersed, as previously described (12). Isolated cells were then transferred into culture medium supplemented with 5 µg/ml insulin, 10 µg/ml apo-transferrin, 20 mg/ml ascorbic acid, and 5% fetal calf serum. Adrenocortical cells were cultured in petri dishes (at a density of 2 x 106 cells/ml) and incubated at 37 C in a 5% CO2-95% air atmosphere with 100% relative humidity. The culture medium was changed 24 h after plating. After 2 d in culture, cells were incubated for 24 h with fresh DMEM (control) or DMEM containing different concentrations of 5-HT, cisapride, hCG, TRH, GnRH, GHRH, ACTH, or glucagon. Then aliquots of culture medium were taken and immediately frozen at –20 C until cortisol RIA. Results are expressed as mean ± SE, and statistical significance was assessed by Bonferroni’s test after one-way ANOVA.
Perifusion experiments. Tumor tissues obtained at surgery from the two patients were studied by using a perifusion system technique as previously described (1). Briefly, tumor fragments were diced into small pieces (1–2 mm3), mixed with biogel P2, and transferred into perifusion chambers. Tumor slices were perifused with DMEM at constant flow rate (300 µl/min) and temperature (37 C). The perifusion medium was continuously gassed with a 95% O2-5% CO2 mixture. The tissues were allowed to stabilize for 2 h before any test substance was administered. Test compounds were dissolved in gassed DMEM and infused into the perifusion chambers at the same flow rate as DMEM alone by means of a multichannel peristaltic pump. Fractions of the effluent perifusate were collected every 5 min and immediately frozen until assay. Cortisol levels were determined in all fractions by RIA, as previously described (1).
Real-time PCR.
Total RNA from the two tumors and seven normal adrenal glands was extracted by the acid guanidium-thiocyanate-phenol-chloroform procedure by using Tri Reagent (Sigma, St. Louis, MO). The concentration of total RNA was determined by measuring the OD at 260 nm. Real-time RT-PCR assays were carried out as described by Fink et al. (13) to quantify 5-HT4 receptor mRNA in both adrenocortical adenomas and normal adrenocortical tissue. The primers and fluorogenic TaqMan probe used for these experiments hybridized to all 5-HT4 receptor splice variants (5-HT4pan, Table 1
). Briefly, 1 µg of total RNA from each tissue was converted to single-stranded cDNA using SuperScript II (Life Technologies Eragny, France) with oligo (dT)12–18 primer (0.5 µg/ml). The cDNA prepared was diluted and aliquoted into microtiter plates. For each 25-µl TaqMan reaction, 5 µl cDNA were mixed with 1 µl water, 12.5 µl TaqMan universal PCR master mix 2X (Applied Biosystems, Courtaboeuf, France), 2 µl sense primer (2 µM), 2 µl antisense primer (2 µM), and 2.5 µl TaqMan probe (2 µM). PCR parameters were 50 C for 2 min, 95 C for 10 min, 40 cycles of 95 C for 15 sec, and 60 C for 1 min. In addition, parallel assays using the same cDNA pools were carried out using primers and probe to the housekeeping gene porphobilinogen deaminase (PBGD, Table 1). Quantitative RT-PCR was performed using an ABI Prism 7700 sequence detector system (Applied Biosystems) and analyzed using relative expression to PBGD, as previously described (13). Briefly, the level of expression in each sample was normalized by dividing copies per nanogram total RNA of 5-HT4 receptor gene by copies per nanogram total RNA of the housekeeping gene PBGD and expressed as a percentage. This mode of calculation allows to correct for both RNA quality and quantity. In each group of tissues, i.e. normal and adenomas, data are presented as mean ± SEM.
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Results |
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In the two patients, plasma ACTH levels remained suppressed [<1 pmol/liter (5 pg/ml)] throughout the in vivo studies (data not shown), indicating that all plasma cortisol responses observed were not mediated by ACTH. Administration of the 5-HT4 receptor agonist cisapride (10 mg, orally) induced a significant increase in plasma cortisol levels reaching +129% in patient 1 and +58% in patient 2 (Fig. 1). A significant stimulation of cortisol secretion was also induced by the glucagon (+25%) and the combined TRH/GnRH/GHRH tests (+42%) in patient 1, terlipressin (+58%) in patient 2, and cosyntropin in the two patients (+542% in patient 1 and +733% in patient 2). None of the other tests induced any significant increase in plasma cortisol levels.
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Effect of 5-HT on cortisol secretion.
Incubation of cultured cells derived from the two tumors with increasing concentrations of 5-HT (10–9 to 10–5 M) provoked a dose-dependent stimulation of cortisol secretion (Fig. 2). 5-HT was less efficient to stimulate steroidogenesis in patient 1 tumor cells (T1: Emax +36.5 ± 2.2%) than normal adrenocortical cells (Emax +124 ± 11%) and patient 2 tumor cells (T2: Emax +271 ± 6%). However, the potency of 5-HT to stimulate cortisol production was higher in T1 (pEC50 8.94 ± 0.02) than normal (pEC50 7.39 ± 0.23) and T2 (pEC50 7.26 ± 0.06) cells. In T1 cells, the corticotropic effect of 5-HT was inhibited by both the specific 5-HT4 receptor antagonist GR 113808 (0.1 µM, pKB 8.86) and the nonspecific serotonergic antagonist methiothepin (0.1 µM, pKB > 10) that exhibits no affinity for the 5-HT4 receptor (Fig. 3). As expected, GR 113808 totally suppressed 5-HT-induced steroidogenesis in normal adrenocortical cells, whereas methiothepin had no influence on the cortisol response to 5-HT (Fig. 3, inset). In T2 cells, 5-HT-induced steroidogenesis was also inhibited by GR 113808 (0.1 µM) and methiothepin (0.1 µM) with pKB values of 7.35 and greater than 9, respectively (Fig. 4A). In these cells, cisapride weakly stimulated cortisol secretion in a dose-dependent manner (Emax +25.5 ± 5.4%) (Fig. 4B), and the response to cisapride was totally abolished by GR 113808 (0.1 µM) but was not influenced by methiothepin (0.1 µM, Fig. 4B). The protein kinase A (PKA) inhibitor H89 (1 µM) significantly reduced the efficacy of 5-HT (Emax +167 ± 14 vs. +271 ± 6% in control conditions, P < 0.0001) to stimulate cortisol production without affecting its potency (pEC50 7.34 ± 0.22) (Fig. 5).
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) and +51% in tumor 2 (Fig. 6B) followed by a gradual decline in the secretion rate. Comparison of the kinetics of the adenoma secretory responses to 5-HT with that previously observed in normal adrenal glands (1) showed that the cortisol response to 5-HT was more prolonged in perifused tumor explants (Fig. 6, A and B).
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Effects of peptide hormones on cortisol secretion from tumor 1 cells.
The corticotropic effects of several peptide hormones were evaluated in cells obtained from patient 1 who had responded in vivo to the glucagon and combined TRH/GHRH/GnRH stimulatory tests. Incubation of cultured cells with graded concentrations of glucagon (10–11 to 10–7 M) induced a significant increase in cortisol secretion (Fig. 7A). The maximum effect of glucagon (+28.7 ± 5.6%) was obtained at a concentration of 10–10 M, and attenuation of the response was observed at higher concentrations (10–9 to 10–7 M). In contrast, glucagon had no effect on the secretory activity of normal adrenocortical cells at concentrations up to 10–7 M (Fig. 7A). As expected, ACTH elicited a robust increase (+128 ± 6.9%) in cortisol production by T1 cells (Fig. 7B). TRH (0.1 µM), GHRH (0.1 µM), and hCG (0.1 µM) had no effect on cortisol secretion. Conversely, GnRH (0.1 µM) induced a +32.7 ± 6.0% increase in steroid output (Fig. 7B) but did not modify cortisol production from normal cells (Fig. 7B, inset).
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, A and B). 5-HT-positive cells had the morphological characteristics of spongiocytic cells, i.e. cells with abundant cytoplasm and numerous lipid droplets (Fig. 8, C and D). Preincubation of the 5-HT antiserum with 5-HT (10–6 M) totally abolished immunostaining (data not shown).
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Discussion |
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To investigate the type(s) of receptor mediating the action of 5-HT on the two adenoma tissues, cultured tumor cells were stimulated by 5-HT in the presence of serotonergic antagonists. In T1 and T2 cells, the corticotropic effect of 5-HT was inhibited by the specific 5-HT4 receptor antagonist GR 113808 as well as methiothepin, which exhibited the highest antagonistic potency. Methiothepin acts as an antagonist on a wide variety of serotonergic receptors including, i.e. the 5-HT1, 5-HT2, 5-HT5, 5-HT6, and 5-HT7 subtypes but has no affinity for the 5-HT4 receptor (4). In agreement with this latter observation, we found that methiothepin had no influence on 5-HT-induced cortisol secretion in normal adrenocortical cells at concentrations up to 10–7 M. These data show therefore that the corticotropic action of 5-HT on the two tumor tissues are mediated by both the eutopic 5-HT4 receptor and an ectopic 5-HT receptor belonging either to the 5-HT1, 5-HT2, 5-HT5, 5-HT6, or 5-HT7 types. However, this observation may not explain the abnormal plasma cortisol responses to cisapride obtained in the two patients for the following reasons: 1) cisapride is unable to activate other serotonergic receptor types than the 5-HT4 receptor (4); 2) 5-HT4 receptor mRNA was not significantly overexpressed in tumor 1 tissue; and 3) although high amounts of 5-HT4 receptor mRNAs were detected by real-time PCR in tumor 2 cells, cisapride weakly stimulated cortisol secretion and GR 113808 inhibited the corticotropic effect of 5-HT with a low potency, indicating that the tissue contained low levels of functional 5-HT4 receptors. Abnormal plasma cortisol responses to cisapride in the two patients may rather involve nonserotonergic mechanisms. In the gastrointestinal tract, 5-HT4 receptor agonists exert in part their prokinetic effects through activation of cholinergic and tachykininergic pathways (17, 18). In the adrenal gland, acetylcholine and tachykinins, which are locally released by splanchnic nerve endings, are able to stimulate glucocorticoid secretion from adrenocortical cells (19, 20, 21, 22). In addition, atropine has been shown to partially inhibit the in vivo stimulatory effect of the 5-HT4 receptor agonist metoclopramide on aldosterone secretion (23). It is thus conceivable that the increased in vivo sensitivity of the two tumors to cisapride administration could be the consequence of enhanced intraadrenal cholinergic and/or tachykininergic tone. It is also possible that cisapride may bind to an unknown 5-HT receptor or a mutated receptor.
In tumor 2 cells, the efficacy of 5-HT to stimulate cortisol secretion was markedly reduced by the PKA inhibitor H89. This observation indicates that the steroidogenic effect of 5-HT in the tumor tissue was mediated by the cAMP pathway. Because activating mutations of the Gs
protein have been shown to induce adrenal hyperplasia, it appears that stimulation of the cAMP pathway can promote adrenocortical cell proliferation (24). Consequently, it is possible that 5-HT may have played a significant role in adrenal tumorigenesis in patient 2. Considering the low physiological plasma concentrations of 5-HT (<10–9 M) (25), it is unlikely that circulating 5-HT may influence cortisol production from the tumor tissues. Therefore, if 5-HT were to play a significant role in the maintenance of active steroidogenesis in the tumors, the indolamine would have to be produced locally. Indeed, immunohistochemical labeling revealed the presence of 5-HT-immunoreactive cells within the tumor tissues, suggesting that cortisol production may actually be under the control of an intratumoral serotonergic tone involving an autocrine/paracrine mode of regulation. Contrary to the normal adrenal cortex in which 5-HT is exclusively stored in perivascular mast cells (1, 3), the tumor tissues exhibited 5-HT-like immunoreactivity in a subpopulation of steroidogenic cells. Thus, it appears that the localization of 5-HT can be ectopic in some adrenocortical tumor tissues.
We previously observed that glucagon has no effect on cortisol secretion in healthy volunteers pretreated with dexamethasone (11). In the present study, clinical testing revealed that glucagon administration induced a +25% increase in plasma cortisol level in patient 1. The fact that plasma ACTH concentration remained suppressed throughout the study suggested that glucagon-evoked cortisol secretion resulted from a direct stimulatory effect of the peptide on adrenal tumor cells. In agreement with this hypothesis, in vitro studies showed that low concentrations of glucagon (10–10 to 10–9 M) stimulated cortisol production from cultured tumor 1 cells. At higher concentrations (10–8 to 10–7 M), glucagon was less efficient to stimulate cortisol secretion, suggesting the occurrence of a desensitization mechanism of the receptor, as formerly demonstrated in rat hepatocytes (26). Consistent with a previous study (27), we did not notice any effect of glucagon on cortisol secretion from normal adrenocortical cells. The present data represent the first demonstration of a cortisol-producing tumor abnormally sensitive in vivo and in vitro to glucagon.
A significant increase in plasma cortisol levels after the combined TRH/GHRH/GnRH stimulatory test was also observed in patient 1, suggesting that the tumor tissue exhibited abnormal sensitivity to TRH, GHRH or GnRH. Aberrant expression of the LH/hCG receptor by adrenocortical tumor cells may also account for a possible GnRH-evoked cortisol secretion as previously observed in several cases of AIMAHs causing Cushing’s syndrome (8, 28). The in vitro studies showed that GnRH, but not TRH, GHRH, or hCG, stimulated cortisol production from cultured tumor 1 cells, indicating that the in vivo cortisol response to the combined TRH/GHRH/GnRH test can be accounted for by an abnormal sensitivity of the tumor to GnRH. This is the first case of an adrenocortical lesion abnormally sensitive in vivo and in vitro to GnRH.
In conclusion, we have described two adrenocortical cortisol-producing adenomas hypersensitive in vivo and in vitro to serotonergic agonists. In vitro pharmacological studies revealed that the cortisol response to 5-HT was mainly mediated by ectopic serotonergic receptors, whereas real-time PCR analysis demonstrated the occurrence of substantial amounts of mRNAs encoding the eutopic 5-HT4 receptor in tumor tissues. The absence of correlation between real-time PCR and pharmacological data indicates that the sole molecular approaches are not sufficient to determine the receptor types actually involved in the regulation of steroidogenesis in functional adrenocortical tumors. Our results also show for the first time the presence of 5-HT in adrenocortical adenomas, providing the basis for autocrine/paracrine regulations of cortisol secretion within the tissues. Finally, we observed that one of the tumors was abnormally sensitive to glucagon and GnRH, showing that cortisol-secreting tumors can express multiple illegitimate receptors in very much the same way as AIMAHs causing Cushing’s syndrome.
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Acknowledgments |
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Footnotes |
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First Published Online February 10, 2005
1 This work is dedicated to the memory of Vincent Contesse, a brilliant researcher and beloved friend. 
Abbreviations: AIMAH, ACTH-independent macronodular adrenal hyperplasia; CT, computed tomography; H89, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride; hCG, human chorionic gonadotropin; 5-HT, serotonin; N, normal value; PBGD, porphobilinogen deaminase; PKA, protein kinase A; UFC, urinary free cortisol.
Received December 16, 2004.
Accepted January 31, 2005.
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
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).
), T1 tumor cells (A; •), and T2 tumor cells (B; 
) and the nonselective 5-HT receptor antagonist methiothepin (10–7 M; 
