Emerging roles of ER-resident selenoproteins in brain physiology and physiopathology.

The brain has a very high oxygen consumption rate and is particularly sensitive to oxidative stress. It is also the last organ to suffer from a loss of selenium (Se) in case of deficiency. Se is a crucial trace element present in the form of selenocysteine, the 21st proteinogenic amino acid present in selenoproteins, an essential protein family in the brain that participates in redox signaling. Among the most abundant selenoproteins in the brain are glutathione peroxidase 4 (GPX4), which reduces lipid peroxides and prevents ferroptosis, and selenoproteins W, I, F, K, M, O and T. Remarkably, more than half of them are proteins present in the ER and recent studies have shown their involvement in the maintenance of ER homeostasis, glycoprotein folding and quality control, redox balance, ER stress response signaling pathways and Ca2+ homeostasis. However, their molecular functions remain mostly undetermined. The ER is a highly specialized organelle in neurons that maintains the physical continuity of axons over long distances through its continuous distribution from the cell body to the nerve terminals. Alteration of this continuity can lead to degeneration of distal axons and subsequent neuronal death. Elucidation of the function of ER-resident selenoproteins in neuronal pathophysiology may therefore become a new perspective for understanding the pathophysiology of neurological diseases. Here we summarize what is currently known about each of their molecular functions and their impact on the nervous system during development and stress.

The SELENOT mimetic PSELT promotes nerve regeneration by increasing axonal myelination in a facial nerve injury model in female rats.

Peripheral nerve injury (PNI) is frequent and many patients suffer lifelong disabilities in severe cases. Although the peripheral nervous system is able to regenerate, its potential is limited. In this study, we tested in a nerve regeneration model in rat the potential beneficial effect of a short mimetic peptide, named PSELT, which derives from SELENOT, an essential thioredoxin-like selenoprotein endowed with neuroprotective and antioxidant activities. For this purpose, the right facial nerve of female Long-Evans rats was axotomized then bridged with a free femoral vein interposition graft. PSELT (1 µM) was injected into the vein immediately and 48 h after the injury, and the effects observed were compared to those found after an end-to-end suture used as a gold standard treatment. Whisking behavior, electrophysiological potential, and histological analyses were performed 3 months after injury to determine the effects of these treatments. These analyses revealed that PSELT-treated animals exhibit a better motor recovery in terms of protraction amplitude and velocity of vibrissae compared to control and end-sutured nerve animal groups. Moreover, administration of PSELT following injury enhanced muscle innervation, axonal elongation, and myelination of newly formed nerve fibers. Altogether, these results indicate that a PSELT-based treatment is sufficient to enhance facial nerve myelination and regeneration and could represent a new therapeutic tool to treat PNI.

Selenoprotein T: An Essential Oxidoreductase Serving as a Guardian of Endoplasmic Reticulum Homeostasis.

Significance: Selenoproteins incorporate the essential nutrient selenium into their polypeptide chain. Seven members of this family reside in the endoplasmic reticulum (ER), the exact function of most of which is poorly understood. Especially, how ER-resident selenoproteins control the ER redox and ionic environment is largely unknown. Since alteration of ER function is observed in many diseases, the elucidation of the role of selenoproteins could enhance our understanding of the mechanisms involved in ER homeostasis. Recent Advances: Among selenoproteins, selenoprotein T (SELENOT) is remarkable as the most evolutionarily conserved and the only ER-resident selenoprotein whose gene knockout in mouse is lethal. Recent data indicate that SELENOT contributes to ER homeostasis: reduced expression of SELENOT in transgenic cell and animal models promotes accumulation of reactive oxygen and nitrogen species, depletion of calcium stores, activation of the unfolded protein response and impaired hormone secretion. Critical Issues: SELENOT is anchored to the ER membrane and associated with the oligosaccharyltransferase complex, suggesting that it regulates the early steps of N-glycosylation. Furthermore, it exerts a selenosulfide oxidoreductase activity carried by its thioredoxin-like domain. However, the physiological role of the redox activity of SELENOT is not fully understood. Likewise, the nature of its redox partners needs to be further characterized. Future Directions: Given the impact of ER stress in pathologies such as neurodegenerative, cardiovascular, metabolic and immune diseases, understanding the role of SELENOT and developing derived therapeutic tools such as selenopeptides to improve ER proteostasis and prevent ER stress could contribute to a better management of these diseases.

Chromogranin A preferential interaction with Golgi phosphatidic acid induces membrane deformation and contributes to secretory granule biogenesis.

Chromogranin A (CgA) is a key luminal actor of secretory granule biogenesis at the trans-Golgi network (TGN) level but the molecular mechanisms involved remain obscure. Here, we investigated the possibility that CgA acts synergistically with specific membrane lipids to trigger secretory granule formation. We show that CgA preferentially interacts with the anionic glycerophospholipid phosphatidic acid (PA). In accordance, bioinformatic analysis predicted a PA-binding domain (PABD) in CgA sequence that effectively bound PA (36:1) or PA (40:6) in membrane models. We identified PA (36:1) and PA (40:6) as predominant species in Golgi and granule membranes of secretory cells, and we found that CgA interaction with these PA species promotes artificial membrane deformation and remodeling. Furthermore, we demonstrated that disruption of either CgA PABD or phospholipase D (PLD) activity significantly alters secretory granule formation in secretory cells. Our findings show for the first time the ability of CgA to interact with PLD-generated PA, which allows membrane remodeling and curvature, key processes necessary to initiate secretory granule budding.

AMPK Activation of PGC-1α/NRF-1-Dependent SELENOT Gene Transcription Promotes PACAP-Induced Neuroendocrine Cell Differentiation Through Tolerance to Oxidative Stress.

Several cues including pituitary adenylate cyclase-activating polypeptide (PACAP), which acts through cAMP stimulation, specify the conversion of sympathoadrenal (SA) precursors toward different cell phenotypes by promoting their survival and differentiation. Selenoprotein T (SELENOT) is a PACAP-stimulated ER oxidoreductase that exerts an essential antioxidant activity and whose up-regulation is associated with SA cell differentiation. In the present study, we investigated the transcriptional cascade elicited by PACAP/cAMP to trigger SELENOT gene transcription during the conversion of PC12 cells from SA progenitor-like cells toward a neuroendocrine phenotype. Unexpectedly, we found that PACAP/cAMP recruits the canonical pathway that regulates mitochondrial function in order to elicit SELENOT gene transcription and the consequent antioxidant response during PC12 cell differentiation. This cascade involves LKB1-mediated AMPK activation in order to stimulate SELENOT gene transcription through the PGC1-α/NRF-1 complex, thus allowing SELENOT to promote PACAP-stimulated neuroendocrine cell survival and differentiation. Our data reveal that a PACAP and cAMP-activated AMPK-PGC-1α/NRF-1 cascade is critical for the coupling of oxidative stress tolerance, via SELENOT gene expression, and mitochondrial biogenesis in order to achieve PC12 cell differentiation. The data further highlight the essential role of SELENOT in cell metabolism during differentiation.

Selenoprotein T is a novel OST subunit that regulates UPR signaling and hormone secretion.

Selenoprotein T (SelT) is a recently characterized thioredoxin-like protein whose expression is very high during development, but is confined to endocrine tissues in adulthood where its function is unknown. We report here that SelT is required for adaptation to the stressful conditions of high hormone level production in endocrine cells. Using immunofluorescence and TEM immunogold approaches, we find that SelT is expressed at the endoplasmic reticulum membrane in all hormone-producing pituitary cell types. SelT knockdown in corticotrope cells promotes unfolded protein response (UPR) and ER stress and lowers endoplasmic reticulum-associated protein degradation (ERAD) and hormone production. Using a screen in yeast for SelT-membrane protein interactions, we sort keratinocyte-associated protein 2 (KCP2), a subunit of the protein complex oligosaccharyltransferase (OST). In fact, SelT interacts not only with KCP2 but also with other subunits of the A-type OST complex which are depleted after SelT knockdown leading to POMC N-glycosylation defects. This study identifies SelT as a novel subunit of the A-type OST complex, indispensable for its integrity and for ER homeostasis, and exerting a pivotal adaptive function that allows endocrine cells to properly achieve the maturation and secretion of hormones.

CRISPR-Barcoding for Intratumor Genetic Heterogeneity Modeling and Functional Analysis of Oncogenic Driver Mutations.

Intratumor genetic heterogeneity underlies the ability of tumors to evolve and adapt to different environmental conditions. Using CRISPR/Cas9 technology and specific DNA barcodes, we devised a strategy to recapitulate and trace the emergence of subpopulations of cancer cells containing a mutation of interest. We used this approach to model different mechanisms of lung cancer cell resistance to EGFR inhibitors and to assess effects of combined drug therapies. By overcoming intrinsic limitations of current approaches, CRISPR-barcoding also enables investigation of most types of genetic modifications, including repair of oncogenic driver mutations. Finally, we used highly complex barcodes inserted at a specific genome location as a means of simultaneously tracing the fates of many thousands of genetically labeled cancer cells. CRISPR-barcoding is a straightforward and highly flexible method that should greatly facilitate the functional investigation of specific mutations, in a context that closely mimics the complexity of cancer.

Selenoprotein T Exerts an Essential Oxidoreductase Activity That Protects Dopaminergic Neurons in Mouse Models of Parkinson's Disease.

AIMS: Oxidative stress is central to the pathogenesis of Parkinson's disease (PD), but the mechanisms involved in the control of this stress in dopaminergic cells are not fully understood. There is increasing evidence that selenoproteins play a central role in the control of redox homeostasis and cell defense, but the precise contribution of members of this family of proteins during the course of neurodegenerative diseases is still elusive. RESULTS: We demonstrated first that selenoprotein T (SelT) whose gene disruption is lethal during embryogenesis, exerts a potent oxidoreductase activity. In the SH-SY5Y cell model of dopaminergic neurons, both silencing and overexpression of SelT affected oxidative stress and cell survival. Treatment with PD-inducing neurotoxins such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or rotenone triggered SelT expression in the nigrostriatal pathway of wild-type mice, but provoked rapid and severe parkinsonian-like motor defects in conditional brain SelT-deficient mice. This motor impairment was associated with marked oxidative stress and neurodegeneration and decreased tyrosine hydroxylase activity and dopamine levels in the nigrostriatal system. Finally, in PD patients, we report that SelT is tremendously increased in the caudate putamen tissue. INNOVATION: These results reveal the activity of a novel selenoprotein enzyme that protects dopaminergic neurons against oxidative stress and prevents early and severe movement impairment in animal models of PD. CONCLUSIONS: Our findings indicate that selenoproteins such as SelT play a crucial role in the protection of dopaminergic neurons against oxidative stress and cell death, providing insight into the molecular underpinnings of this stress in PD.

Concordant localization of functional urotensin II and urotensin II-related peptide binding sites in the rat brain: Atypical occurrence close to the fourth ventricle.

Urotensin II (UII) and Urotensin II-related peptide (URP) are structurally related paralog peptides that exert peripheral and central effects. UII binding sites have been partly described in brain, and those of URP have never been reported. We exhaustively compared [(125)I]-UII and -URP binding site distributions in the adult rat brain, and found that they fully overlapped at the regional level. We observed UII/URP binding sites in structures lining ventricles, comprising the sphenoid nucleus and cell rafts scattered on a line joining the fourth ventricle and its lateral recess. After injection of UII and URP in the lateral ventricle, we observed c-Fos-positive cell nuclei in areas close to the fourth ventricle, indicating that these receptors are functional. Different c-Fos-containing cell populations were activated. They were all positive for vimentin and glial fibrillary acidic protein (GFAP), excluding the possibility of an ependymal nature. In conclusion, this study demonstrated that UII and URP binding sites are totally overlapping and that these sites were functional in regions bordering the fourth ventricle. These data support a role for UII/URP at the interface between brain parenchyma and cerebrospinal fluid.

The PACAP-regulated gene selenoprotein T is abundantly expressed in mouse and human ß-cells and its targeted inactivation impairs glucose tolerance.

Selenoproteins are involved in the regulation of redox status, which affects several cellular processes, including cell survival and homeostasis. Considerable interest has arisen recently concerning the role of selenoproteins in the regulation of glucose metabolism. Here, we found that selenoprotein T (SelT), a new thioredoxin-like protein of the endoplasmic reticulum, is present at high levels in human and mouse pancreas as revealed by immunofluorescence and quantitative PCR. Confocal immunohistochemistry studies revealed that SelT is mostly confined to insulin- and somatostatin-producing cells in mouse and human islets. To elucidate the role of SelT in ß-cells, we generated, using a Cre-Lox strategy, a conditional pancreatic ß-cell SelT-knockout C57BL/6J mice (SelT-insKO) in which SelT gene disruption is under the control of the rat insulin promoter Cre gene. Glucose administration revealed that male SelT-insKO mice display impaired glucose tolerance. Although insulin sensitivity was not modified in the mutant mice, the ratio of glucose to insulin was significantly higher in the SelT-insKO mice compared with wild-type littermates, pointing to a deficit in insulin production/secretion in mutant mice. In addition, morphometric analysis showed that islets from SelT-insKO mice were smaller and that their number was significantly increased compared with islets from their wild-type littermates. Finally, we found that SelT is up-regulated by pituitary adenylate cyclase-activating polypeptide (PACAP) in ß-pancreatic cells and that SelT could act by facilitating a feed-forward mechanism to potentiate insulin secretion induced by the neuropeptide. Our findings are the first to show that the PACAP-regulated SelT is localized in pancreatic ß- and δ-cells and is involved in the control of glucose homeostasis.

Metoclopramide stimulates catecholamine- and granin-derived peptide secretion from pheochromocytoma cells through activation of serotonin type 4 (5-HT4) receptors.

UNLABELLED: The gastroprokinetic agent metoclopramide is known to stimulate catecholamine secretion from pheochromocytomas. The aim of the study was to investigate the mechanism of action of metoclopramide and expression of serotonin type 4 (5-HT(4)) receptors in pheochromocytoma tissues. Tissue explants, obtained from 18 pheochromocytomas including the tumor removed from a 46-year-old female patient who experienced life-threatening hypertension crisis after metoclopramide administration and 17 additional pheochromocytomas (9 benign and 8 malignant) were studied. Cultured pheochromocytoma cells derived from the patient who previously received metoclopramide were incubated with metoclopramide and various 5-HT(4) receptor ligands. In addition, total mRNAs were extracted from all the 18 tumors. Catecholamine- and granin-derived peptide concentrations were measured in pheochromocytoma cell incubation medium by HPLC and radioimmunological assays. In addition, expression of 5-HT(4) receptor mRNAs in the 18 pheochromocytomas was investigated by the use of reverse transcriptase-PCR. RESULTS: Metoclopramide and the 5-HT(4) receptor agonist cisapride were found to activate catecholamine- and granin-derived peptide secretions by cultured tumor cells. Metoclopramide- and cisapride-evoked catecholamine- and granin-derived peptide productions were inhibited by the 5-HT(4) receptor antagonist GR 113808. 5-HT(4) receptor mRNAs were detected in the patient's tumor and the series of 17 additional pheochromocytomas. This study shows that pheochromocytomas express functional 5-HT(4) receptors that are responsible for the stimulatory action of metoclopramide on catecholamine- and granin-derived peptide secretion. All 5-HT(4) receptor agonists must therefore be contraindicated in patients with proven or suspected pheochromocytoma.

Characterization of urotensin II, distribution of urotensin II, urotensin II-related peptide and UT receptor mRNAs in mouse: evidence of urotensin II at the neuromuscular junction.

Urotensin II (UII) and UII-related peptide (URP) are paralog neuropeptides whose existence and distribution in mouse have not yet been investigated. In this study, we showed by HPLC/RIA analysis that the UII-immunoreactive molecule in the mouse brain corresponds to a new UII(17) isoform. Moreover, calcium mobilization assays indicated that UII(17) and URP were equally potent in stimulating UII receptor (UT receptor). Quantitative RT-PCR and in situ hybridization analysis revealed that in the CNS UII and URP mRNAs were predominantly expressed in brainstem and spinal motoneurons. Besides, they were differentially expressed in the medial vestibular nucleus, locus coeruleus and the ventral medulla. In periphery, both mRNAs were expressed in skeletal muscle, testis, vagina, stomach, and gall bladder, whereas only URP mRNA could be detected in the seminal vesicle, heart, colon, and thymus. By contrast, the UT receptor mRNA was widely expressed, and notably, very high amounts of transcript occurred in skeletal muscle and prostate. In the biceps femoris muscle, UII-like immunoreactivity was shown to coexist with synaptophysin in muscle motor end plate regions. Altogether these results suggest that (i) UII and URP may have many redundant biological effects, especially at the neuromuscular junction; (ii) URP may more specifically participate to autonomic, cardiovascular and reproductive functions.

Expression of serotonin7 receptor and coupling of ectopic receptors to protein kinase A and ionic currents in adrenocorticotropin-independent macronodular adrenal hyperplasia causing Cushing's syndrome.

CONTEXT: In ACTH-independent macronodular adrenal hyperplasia (AIMAH) causing Cushing's syndrome, cortisol secretion is controlled by illegitimate membrane receptors. OBJECTIVE: The aim of the present study was to characterize the pharmacological properties and the transduction mechanisms of illegitimate receptors, i.e. receptors for serotonin (5-HT), gastric inhibitory polypeptide (GIP), and LH/human chorionic gonadotropin (hCG), expressed by AIMAH tissues to evaluate the role of ectopic receptors in the physiopathology of Cushing's syndrome. DESIGN: We used in vitro studies on cultured adrenal hyperplasia cells. SETTING: The setting was a university research laboratory. PATIENTS: AIMAH tissues (H1-H3) were removed from three patients previously screened for illegitimate receptors. MAIN OUTCOME MEASURE(S): The main outcome measures were steroidogenic and electrical activities of cultured adrenal hyperplasia cells. RESULTS: In vitro studies showed that the corticotropic effect of 5-HT was mediated by ectopic 5-HT7 receptors in H1 and H2. GIP and hCG stimulated cortisol production via activation of cAMP-dependent protein kinase A in H2. On the contrary, the protein kinase A inhibitor H-89 did not affect hCG-induced cortisol production in H3. Activation of 5-HT7 or GIP receptors enhanced T-type calcium current in H1 or H2 and H3, respectively. In addition, GIP reduced the amplitude of transient and sustained potassium currents in H2. Conversely, hCG did not modify T-type calcium current in H3. CONCLUSIONS: These data show that, besides their coupling to the cAMP pathway, illegitimate adrenal receptors can activate additional transduction mechanisms, including modulation of membrane channels.

The N-terminal neurotensin fragment, NT1-11, inhibits cortisol secretion by human adrenocortical cells.

CONTEXT: Neurotensin (NT) modulates corticosteroid secretion from the mammalian adrenal gland. OBJECTIVE: The objective of this study was to investigate the possible involvement of NT in the control of cortisol secretion in the human adrenal gland. DESIGN: In vitro studies were conducted on cultured human adrenocortical cells. SETTING: This study was conducted in a university research laboratory. PATIENTS: Adrenal explants from patients undergoing expanded nephrectomy for kidney cancer were studied. MAIN OUTCOME MEASURE: Cortisol secretion from cultured adrenocortical cells was measured. RESULTS: NT1-11, the N-terminal fragment of NT, dose-dependently inhibited basal and ACTH-stimulated cortisol production by human adrenocortical cells in primary culture. In contrast, NT had no influence on cortisol output at concentrations up to 10(-6) m. HPLC and RT-PCR analyses failed to detect any significant amounts of NT and NT mRNA, respectively, in adrenal extracts. Molecular and pharmacological studies were performed to determine the type of NT receptor involved in the corticostatic effect of NT1-11. RT-PCR analysis revealed the expression of NT receptor type (NTR) 3 mRNA but not NTR1 and NTR2 mRNAs in the human adrenal tissue. However, the pharmacological profile of the adrenal NT1-11 receptor was different from that of NTR3, indicating that this receptor type is not involved in the action of NT1-11 on corticosteroidogenesis. CONCLUSION: Our results indicate that NT1-11 may act as an endocrine factor to inhibit cortisol secretion through activation of a receptor distinct from the classical NTR1, NTR2, and NTR3.

Localization and characterization of pituitary adenylate cyclase-activating polypeptide receptors in the human cerebellum during development.

Pituitary adenylate cyclase-activating polypeptide (PACAP) receptors are actively expressed in the cortical layers of the cerebellum of rodents and contribute to cerebellar development. The present report provides the first anatomical localization and characterization of PACAP receptors in the developing human cerebellum. RT-PCR analysis from 15-week-old fetuses to 22-year-old subject showed that PAC1-R and VPAC1-R are expressed in the cerebellum at all stages, whereas VPAC2-R mRNA was barely detectable. In situ hybridization labeling indicated that, in human fetuses, PAC1-R mRNA is associated with the external granule cell layer (EGL), a germinative neuroepithelium, and with the internal granule cell layer (IGL). The distribution pattern of VPAC1-R mRNA was very similar to that of PAC1-R mRNA, whereas VPAC2-R mRNA was visualized only in 7-22-year-old subjects. The localization of [(125)I]PACAP27 binding sites was fully consistent with the distribution of PAC1-R and VPAC1-R mRNA. Pharmacological characterization revealed that, in the EGL and IGL from 15-24-week-old fetuses and in the granule cell layer from 7-22-year-old patients, binding sites exhibit a PAC1-R profile. In contrast, PACAP binding sites observed in the molecular layer and medulla of the adult cerebellum consisted of a heterogeneous population of PAC1-R and VPAC(1/2)-R. Altogether, these data provide the first evidence that PACAP receptors are expressed in the human cerebellar cortex. PAC1-R is the predominant PACAP receptor found in fetuses, and both PAC1-R and VPAC1-R are expressed in the mature cerebellum. These observations suggest that PACAP has neurodevelopmental functions in the human cerebellum.

Localization of the urotensin II receptor in the rat central nervous system.

The vasoactive peptide urotensin II (UII) is primarily expressed in motoneurons of the brainstem and spinal cord. Intracerebroventricular injection of UII provokes various behavioral, cardiovascular, motor, and endocrine responses in the rat, but the distribution of the UII receptor in the central nervous system (CNS) has not yet been determined. In the present study, we have investigated the localization of UII receptor (GPR14) mRNA and UII binding sites in the rat CNS. RT-PCR analysis revealed that the highest density of GPR14 mRNA occurred in the pontine nuclei. In situ hybridization histochemistry showed that the GPR14 gene is widely expressed in the brain and spinal cord. In particular, a strong hybridization signal was observed in the olfactory system, hippocampus, olfactory and medial amygdala, hypothalamus, epithalamus, several tegmental nuclei, locus coeruleus, pontine nuclei, motor nuclei, nucleus of the solitary tract, dorsal motor nucleus of the vagus, inferior olive, cerebellum, and spinal cord. Autoradiographic labeling of brain slices with radioiodinated UII showed the presence of UII-binding sites in the lateral septum, bed nucleus of the stria terminalis, medial amygdaloid nucleus, anteroventral thalamus, anterior pretectal nucleus, pedunculopontine tegmental nucleus, pontine nuclei, geniculate nuclei, parabigeminal nucleus, dorsal endopiriform nucleus, and cerebellar cortex. Intense expression of the GPR14 gene in some hypothalamic nuclei (supraoptic, paraventricular, ventromedian, and arcuate nuclei), in limbic structures (amygdala and hippocampus), in medullary nuclei (solitary tract, dorsal motor nucleus of the vagus), and in motor control regions (cerebral and cerebellar cortex, substantia nigra, pontine nuclei) provides the anatomical substrate for the central effects of UII on behavioral, cardiovascular, neuroendocrine, and motor functions. The occurrence of GPR14 mRNA in cranial and spinal motoneurons is consistent with the reported autocrine/paracrine action of UII on motoneurons.

Expression profile of serotonin4 (5-HT4) receptors in adrenocortical aldosterone-producing adenomas.

OBJECTIVE: We aimed to investigate the expression profile of serotonin4 (5-HT4) receptors in adrenocortical aldosterone-producing adenoma (APA) tissues in comparison with normal adrenal cortex. DESIGN AND METHODS: Total 5-HT4 receptor mRNAs were quantified by real-time quantitative polymerase chain reaction (PCR) assay, and the mRNAs encoding the 5-HT4 receptor isoforms were characterized by reverse transcription (RT)-PCR in seven normal adrenal cortices and 11 APA tissues. The distribution of 5-HT4 receptor mRNAs was investigated by in situ hybridization in both normal adrenal and APA tissues, and the presence of 5-HT in APA tissues was studied by immunohistochemistry. RESULTS: Real-time PCR analysis revealed that 5-HT4 receptor mRNA expression was 4.7-47 times higher in APA tissues than in normal glands. In situ hybridization studies showed that 5-HT4 receptor mRNAs were expressed in both zona glomerulosa and zona fasciculata/reticularis of the normal cortex and in groups of APA steroidogenic cells disseminated in the tumor tissues. Characterization of 5-HT4 receptor splice variants by RT-PCR revealed different profiles of expression in APAs versus normal adrenals. Isoforms (a) and (b) were not expressed in any APA but were present in the majority of normal adrenocortical tissues. Conversely, isoform (d) was expressed in 5/11 APAs but only in 1/7 adrenals. Immunohistochemical studies revealed the presence of 5-HT-immunoreactivity in both mast cells and clusters of steroidogenic cells in APA tissues. CONCLUSION: Our results show overexpression and different splicing of the 5-HT4 receptor in APA tissues in comparison with normal adrenocortical tissue. They also demonstrate the presence of 5-HT in both mast cells and tumor steroidogenic cells, providing evidence for a possible autocrine/paracrine activation of aldosterone secretion within adenoma tissues.

Abnormal sensitivity of cortisol-producing adrenocortical adenomas to serotonin: in vivo and in vitro studies.

Two patients with incidentally discovered adrenocortical adenomas underwent a series of pharmacological and physiological tests after pretreatment with dexamethasone. Illicit plasma cortisol responses to the serotonin (5-HT)4 receptor agonist cisapride were observed in the two patients. Significant increases in plasma cortisol levels were also noticed after glucagon and combined TRH/GnRH/GHRH stimulation tests in patient 1 and after administration of the lysine vasopressin precursor terlipressin in patient 2. After adrenalectomy, in vitro studies were conducted to investigate the cortisol responses of cultured tumor cells to serotonergic ligands and peptide hormones. In the two cases, 5-HT stimulated cortisol secretion from tumor cells with increased efficacy and/or potency to activate steroidogenesis by comparison with normal adrenocortical cells. The corticotropic effect of 5-HT was inhibited by the specific 5-HT4 receptor antagonist GR 113808 and more potently by methiothepin, a nonspecific serotonergic antagonist having no affinity for the 5-HT4 receptor. These results show that the hypersensitivity of the tumors to 5-HT was related to tissue expression of an ectopic serotonergic receptor in addition to the eutopic 5-HT4 receptor. In the two adenoma tissues, immunohistochemical studies revealed the presence of 5-HT-like immunoreactivity within clusters of steroidogenic cells, suggesting that 5-HT acted through an autocrine/paracrine mechanism to stimulate steroidogenesis. Glucagon and GnRH but not TRH, GHRH, and human chorionic gonadotropin stimulated cortisol secretion from tumor 1 cells. In conclusion, this study provides the first observation of adrenocortical cortisol-producing adenomas hypersensitive in vivo and in vitro to serotonergic agonists. Our results also show that cortisol-producing adenomas can express simultaneously several illegitimate receptors.

Overexpression of serotonin4 receptors in cisapride-responsive adrenocorticotropin-independent bilateral macronodular adrenal hyperplasia causing Cushing's syndrome.

The serotonin4 (5-HT4) receptor agonists cisapride and/or metoclopramide have been shown to stimulate cortisol secretion in some patients with ACTH-independent bilateral macronodular adrenal hyperplasias (AIMAH) causing Cushing's syndrome. In the present study, we have investigated quantitatively and qualitatively the expression of the 5-HT4 receptor in both normal adrenal cortex and tissues removed from six patients (P1-P6) with cisapride-responsive AIMAH and Cushing's syndrome. Real-time quantitative PCR assay revealed that the 5-HT4 receptor was overexpressed in four of the six hyperplasias studied when compared with normal adrenal cortex. In these tissues, 5-HT4 receptor mRNA expression was 3 to 16 times higher than in normal glands, likely explaining the abnormal in vivo cortisol response to cisapride. Characterization of 5-HT4 receptor splice variants by RT-PCR in both hyperplastic and normal adrenals showed that the variants present in the two hyperplasias that did not overexpress the 5-HT4 receptor, i.e. P2 and P5, could also be detected in the normal adrenal tissue. In addition, sequencing of the full-length cDNAs encoding 5-HT4 receptors in hyperplasias P2 and P5 did not reveal any mutation. Taken together, our results show an overexpression of the 5-HT4 receptor in cisapride-responsive AIMAH. However, in two cases, the level of expression of the receptor in the hyperplastic adrenal cortex was similar to that of normal adrenal gland. The enhanced sensitivity of these two tissues to 5-HT4 receptor agonists was not due to ectopic expression of 5-HT4 receptor isoforms or to the occurrence of somatic gain-of-function mutation of the receptor.

Characterization of serotonin(4) receptors in adrenocortical aldosterone-producing adenomas: in vivo and in vitro studies.

We have previously shown that serotonin (5-HT) stimulates aldosterone secretion from the human adrenal gland through activation of 5-HT(4) receptors. The aim of the present study was to investigate in vivo and in vitro the presence of 5-HT(4) receptors in aldosterone-producing adenomas (aldosteronomas). Eight patients with aldosteronoma received a single oral dose of placebo or cisapride (10 mg). Cisapride administration significantly increased plasma aldosterone within 120 min without any significant change in renin, cortisol, or potassium levels. In two patients, a marked decrease in the plasma aldosterone response to cisapride was observed after surgical removal of the tumor. The effects of 5-HT and selective 5-HT(4) ligands on aldosterone production from aldosteronoma tissues were studied in vitro using a perifusion system technique. 5-HT and the 5-HT(4) receptor agonist cisapride (10(-7) M, 20 min) both stimulated aldosterone secretion from aldosteronoma slices. The 5-HT- and cisapride-evoked aldosterone responses were inhibited by concomitant administration of the specific 5-HT(4) receptor antagonist GR 113808 (10(-7) M, 150 min). PCR amplification revealed the expression of 5-HT(4) receptor mRNA in 13 of 14 aldosteronomas studied. Taken together, these data show that most aldosteronomas, like normal glomerulosa cells, express a functional 5-HT(4) receptor. Our results also suggest that 5-HT, which can be locally released by intratumoral mast cells, may play a role in the pathophysiology of these tumors.