Dorthe Cartier

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. J. Comp. Neurol. 495:21–36, 2006.

Expression of vasopressin receptors in ACTH-independent macronodular bilateral adrenal hyperplasia causing Cushing's syndrome: molecular, immunohistochemical and pharmacological correlates

Cortisol secretion in ACTH-independent macronodular adrenal hyperplasia (AIMAH) causing Cushings syndrome can be controlled by illegitimate receptors. The aim of the present study was to characterize the molecular, immunohistochemical, and pharmacological profiles of vasopressin receptors in cells derived from three patients with AIMAH (H1-H3), in order to evaluate the role of ectopic vasopressin receptors in the physiopathology of hypercortisolism. Expression of mRNAs encoding the vasopressin receptor types (V(1a), V(1b), and V(2)) were analyzed by RT-PCR in adrenal tissues. The presence of V(1a) and V(2) receptors was studied by immunohistochemistry on adrenal sections. The pharmacological profiles of vasopressin receptors involved in the control of cortisol secretion were investigated using the V(1a) receptor antagonist SR49059 and the V(2) receptor agonist [deamino-Cys(1), Val(4), D-Arg(8)]-vasopressin on cultured cells. The V(1a) receptor protein was present and functional in H1 and H3 tissues, whereas the V(1b) receptor was not expressed in any of the tissues. RT-PCR experiments revealed that V(2) receptor mRNAs were detected in the three tissues. In contrast, immunohistochemical and cell incubation studies showed that the V(2) receptor was involved in the stimulatory effect of AVP on cortisol secretion in H1 and H2, but not in H3 cells. Taken together, these data show that expression of functional ectopic V(2) receptors and repression of eutopic V(1a) receptor can coexist in some hyperplastic corticosteroidogenic tissues. They also reveal that immunohistochemical and incubation studies are essential for the characterization of ectopic receptors actually involved in the control of cortisol secretion by AIMAHs.

The PACAP-Regulated Gene Selenoprotein T Is Abundantly Expressed in Mouse and Human β-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 β-cells, we generated, using a Cre-Lox strategy, a conditional pancreatic β-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 β-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 β- and δ-cells and is involved in the control of glucose homeostasis.

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 [125I]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. J. Comp. Neurol. 496:468–478, 2006.

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 UII17 isoform. Moreover, calcium mobilization assays indicated that UII17 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.

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 patients 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.

Paradoxical inhibitory effect of serotonin on cortisol production from adrenocortical lesions causing Cushing's syndrome.

In the human adrenal gland, serotonin (5‐HT) stimulates cortisol production through a paracrine mechanism involving 5‐HT4 receptors positively‐coupled to adenylyl cyclase. A hyperresponsiveness of adrenocortical tissue to 5‐HT has also been described in several cases of ACTH‐independent bilateral macronodular adrenal hyperplasias (AIMAHs) and adenomas causing Cushings syndrome. In the present study, we report two cases of cortisol‐producing adrenocortical lesions, i.e. one AIMAH (case 1) and one adenoma (case 2), whose secretory activity was inhibited in vitro by 5‐HT. The potencies (pIC50) and efficacies (Emax) of 5‐HT to inhibit cortisol secretion were 8.2 ± 0.4 and − 64.1% ± 7.5% in case 1, and 9.2 ± 0.5 and − 32.3% ± 3.8% in case 2. The specific 5‐HT4 antagonist GR 113808 failed to influence the 5‐HT‐induced decrease in cortisol production by the two tissues, indicating that the paradoxical inhibitory effect of 5‐HT could not be accounted for by activation of eutopic 5‐HT4 receptors. These results suggest that the tissues expressed aberrant 5‐HT receptors. In conclusion, the present study provides the first evidence for an inhibitory effect of 5‐HT on cortisol secretion in adrenocortical lesions causing Cushings syndrome. Our data also suggest that expression of illegitimate membrane receptors by cortisol‐producing adrenal hyperplasias and/or adenomas may convert a paracrine stimulatory factor into an inhibitory signal.

Effect of Serotonin4 (5-HT4) Receptor Agonists on Aldosterone Secretion in Idiopathic Hyperaldosteronism

Serotonin (5-HT) stimulates aldosterone secretion in man through 5-HT4 receptors positively coupled to adenylyl cyclase. In particular, it has been shown that oral administration of a single dose of the 5-HT4 receptor agonist cisapride induces a significant increase in plasma aldosterone levels (PAL) in healthy volunteers. Idiopathic hyperaldosteronism (IH) is a rare disorder characterized by hypertension, hypokalemia and bilateral adrenal hypersecretion of aldosterone. In patients with IH, administration of the 5-HT precursor 5-hydroxytryptophan (5-HTP) is followed by a significant increase in PAL. 5-HTP-induced aldosterone secretion has been attributed to the activation of central serotonergic pathways. The aim of the present study was to evaluate the effect of the oral administration of a single dose of cisapride (10 mg) on aldosterone secretion in 15 patients with IH, in a simple blind fashion versus placebo. Cisapride induced a significant increase in PAL but did not affect renin, cortisol and potassium levels. The present study demonstrates that 5-HT4 receptor agonists are able to stimulate aldosterone secretion in patients with IH. These data also indicate that hyperplastic glomerulosa tissue, like normal glomerulosa cells, expresses a functional 5-HT4 receptor. Therefore, 5-HT4 receptor antagonists may represent a new approach in the treatment of primary hyperaldosteronism.

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.

Selenoprotein T: From Discovery to Functional Studies Using Conditional Knockout Mice

Selenoprotein T (SELT) is a thioredoxin-like enzyme that exerts an essential oxidoreductase activity in the endoplasmic reticulum during development and after tissue injury where its expression is highly induced. Disruption of the Selt gene is lethal during embryogenesis, and its conditional knockout in the brain causes the reduction of several cerebral structures and increases the vulnerability of mice to neurotoxin-induced neurodegeneration. While its expression is silenced in most tissues in the adult, SELT persists at high levels in endocrine tissues such as the pancreas where it controls hormone production. Thus, SELT could be involved in the redox circuits that control homeostasis and survival of cells with intense metabolic activity during development or in adult endocrine and lesioned cerebral tissues.