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 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.

Pituitary adenylate cyclase-activating polypeptide (PACAP) promotes both survival and neuritogenesis in PC12 cells through activation of nuclear factor κB (NF-κB) pathway: involvement of extracellular signal-regulated kinase (ERK), calcium, and c-REL.

The pituitary adenylate cyclase-activating polypeptide (PACAP) is a trophic factor that promotes neuronal survival and neurite outgrowth. However, the signaling pathways and the transcriptional mechanisms involved are not completely elucidated. Our previous studies aimed at characterizing the transcriptome of PACAP-differentiated PC12 cells revealed an increase in the expression of nuclear factor κB2 (NF-κB2) gene coding for p100/p52 subunit of NF-κB transcription factor. Here, we examined the role of the NF-κB pathway in neuronal differentiation promoted by PACAP. We first showed that PACAP-driven survival and neuritic extension in PC12 cells are inhibited following NF-κB pathway blockade. PACAP stimulated both c-Rel and p52 NF-κB subunit gene expression and nuclear translocation, whereas c-Rel down-regulation inhibited cell survival and neuritogenesis elicited by the neuropeptide. PACAP-induced c-Rel nuclear translocation was inhibited by ERK1/2 and Ca(2+) blockers. Furthermore, the neuropeptide stimulated NF-κB p100 subunit processing into p52, indicative of activation of the NF-κB alternative pathway. Taken together, our data show that PACAP promotes both survival and neuritogenesis in PC12 cells by activating NF-κB pathway, most likely via classical and alternative signaling cascades involving ERK1/2 kinases, Ca(2+), and c-Rel/p52 dimers.

Impact of gene/genome duplications on the evolution of the urotensin II and somatostatin families.

The present review describes the molecular evolution of two phylogenetically related families of neuropeptides, the urotensin II (UII) and somatatostatin (SS) families. The UII family consists of four paralogous genes called UII, URP, URP1 and URP2 and the SS family is composed of six paralogous genes named SS1, SS2, SS3, SS4, SS5 and SS6. All these paralogs are present in teleosts, while only four of them, UII, URP, SS1 and SS2 are detected in tetrapods. Comparative genomics showed that most of these genes, namely UII, URP, URP1 and URP2 on the one hand and SS1, SS2 and SS5 on the other hand arose through the 2R. In contrast, the teleost-specific 3R had a much more moderate impact since it only concerned the UII and SS1 genes, which once duplicated, generated a second UII copy and SS4, respectively. The two remaining genes, SS3 and SS6, arose through tandem duplications of the SS1 and SS2 genes respectively, probably in the stem lineage of actinopterygians, before the emergence of teleosts. The history of the UII and SS families has also been marked by massive gene lost, both in tetrapods and in teleosts, but only after the 3R in this latter lineage. Finally, ancestral UII and SS genes are thought to have arisen through tandem duplication of a single ancestral gene, largely before the 1R. An important challenge for the future will be to understand the physiological significance of the molecular diversity of these two families.

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.

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.

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.

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.

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.

Comparative distribution of the mRNAs encoding urotensin I and urotensin II in zebrafish.

The neural neurosecretory system of fishes produces two biologically active neuropeptides, i.e. the corticotropin-releasing hormone paralog urotensin I (UI) and the somatostatin-related peptide urotensin II (UII). In zebrafish, we have recently characterized two UII variants termed UIIalpha and UIIbeta. In the present study, we have investigated the distribution of UI, UIIalpha and UIIbeta mRNAs in different organs by quantitative RT-PCR analysis and the cellular localization of the three mRNAs in the spinal cord by in situ hybridization (ISH) histochemistry. The data show that the UI gene is mainly expressed in the caudal portion of the spinal cord and, to a lesser extent, in the brain, while the UIIalpha and the UIIbeta genes are exclusively expressed throughout the spinal cord. Single-ISH labeling revealed that UI, UIIalpha and UIIbeta mRNAs occur in large cells, called Dahlgren cells, located in the ventral part of the caudal spinal cord. Double-ISH staining showed that UI, UIIalpha and UIIbeta mRNAs occur mainly in distinct cells, even though a few cells were found to co-express the UI and UII genes. The differential expression of UI, UIIalpha and UIIbeta genes may contribute to the adaptation of Dahlgren cell activity during development and/or in various physiological conditions.

Selenoprotein T is a key player in ER proteostasis, endocrine homeostasis and neuroprotection.

Selenoprotein T (SELENOT, SELT) is a thioredoxin-like enzyme anchored at the endoplasmic reticulum (ER) membrane, whose primary structure is highly conserved during evolution. SELENOT is abundant in embryonic tissues and its activity is essential during development since its gene knockout in mice is lethal early during embryogenesis. Although its expression is repressed in most adult tissues, SELENOT remains particularly abundant in endocrine organs such as the pituitary, pancreas, thyroid and testis, suggesting an important role of this selenoprotein in hormone production. Our recent studies showed indeed that SELENOT plays a key function in insulin and corticotropin biosynthesis and release by regulating ER proteostasis. Although SELENOT expression is low or undetectable in most cerebral structures, its gene conditional knockout in brain provokes anatomical alterations that impact mice behavior. This suggests that SELENOT also plays an important role in brain development and function. In addition, SELENOT is induced after injury in brain or liver and exerts a cytoprotective effect. Thus, the data gathered during the last ten years of intense investigation of this newly discovered thioredoxin-like enzyme point to an essential function during development and in adult endocrine organs or lesioned brain, most likely by regulating ER redox circuits that control homeostasis and survival of cells with intense metabolic activity.

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.

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.

Structure-activity relationships of a novel series of urotensin II analogues: identification of a urotensin II antagonist.

Urotensin II (U-II) is a potent vasoconstrictor peptide which has been identified as the endogenous ligand for the orphan G protein-coupled receptor GPR14 now renamed UT receptor. As the C-terminal cyclic hexapeptide of U-II (U-II(4-11), H-Asp-Cys-Phe-Trp-Lys-Tyr-Cys-Val-OH) possesses full biological activity, we have synthesized a series of U-II(4-11) analogues and measured their binding affinity on hGPR14-transfected CHO cells and their contractile activity on de-endothelialized rat aortic rings. The data indicate that a free amino group and a functionalized side-chain at the N-terminal extremity of the peptide are not required for biological activity. In addition, the minimal chemical requirement at position 9 of U-II(4-11) is the presence of an aromatic moiety. Most importantly, replacement of the Phe6 residue by cyclohexyl-Ala (Cha) led to an analogue, [Cha6]U-II(4-11), that was devoid of agonistic activity but was able to dose-dependently suppress the vasoconstrictor effect of U-II on rat aortic rings. These new pharmacological data, by providing further information regarding the structure-activity relationships of U-II analogues, should prove useful for the rational design of potent and nonpeptidic UT receptor agonists and antagonists.

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.

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.

Linkage mapping of the [Pro2]somatostatin-14 gene in zebrafish: evolutionary perspectives.

Radiation hybrid mapping assigned the zebrafish [Pro(2)]somatostatin-14 (also termed somatostatin 2; SS2) gene to linkage group 23 of the zebrafish genome, close to the marker nadl1.2. Comparative genomic analysis revealed conserved syntenies of the SS2 gene locus with part of the human 1p36 region, where the cortistatin gene is located. This observation strongly suggests that the SS2 gene in nonmammalian species and the cortistatin gene in mammals are orthologous.

Comparative distribution and in vitro activities of the urotensin II-related peptides URP1 and URP2 in zebrafish: evidence for their colocalization in spinal cerebrospinal fluid-contacting neurons.

Urotensin II (UII) is an evolutionarily conserved neuropeptide initially isolated from teleost fish on the basis of its smooth muscle-contracting activity. Subsequent studies have demonstrated the occurrence of several UII-related peptides (URPs), such that the UII family is now known to include four paralogue genes called UII, URP, URP1 and URP2. These genes probably arose through the two rounds of whole genome duplication that occurred during early vertebrate evolution. URP has been identified both in tetrapods and teleosts. In contrast, URP1 and URP2 have only been observed in ray-finned and cartilaginous fishes, suggesting that both genes were lost in the tetrapod lineage. In the present study, the distribution of urp1 mRNA compared to urp2 mRNA is reported in the central nervous system of zebrafish. In the spinal cord, urp1 and urp2 mRNAs were mainly colocalized in the same cells. These cells were also shown to be GABAergic and express the gene encoding the polycystic kidney disease 2-like 1 (pkd2l1) channel, indicating that they likely correspond to cerebrospinal fluid-contacting neurons. In the hindbrain, urp1-expressing cells were found in the intermediate reticular formation and the glossopharyngeal-vagal motor nerve nuclei. We also showed that synthetic URP1 and URP2 were able to induce intracellular calcium mobilization in human UII receptor (hUT)-transfected CHO cells with similar potencies (pEC50=7.99 and 7.52, respectively) albeit at slightly lower potencies than human UII and mammalian URP (pEC50=9.44 and 8.61, respectively). The functional redundancy of URP1 and URP2 as well as the colocalization of their mRNAs in the spinal cord suggest the robustness of this peptidic system and its physiological importance in zebrafish.

Expression and processing of the [Pro(2),Met(13)]somatostatin-14 precursor in the intermediate lobe of the frog pituitary.

The biosynthesis of various hypothalamic neuropeptides has been previously reported in anterior pituitary cells but not in intermediate lobe cells. We have recently demonstrated the occurrence of two somatostatin isoforms in the frog brain, namely somatostatin-14 (SS1) and [Pro(2),Met(13)]somatostatin-14 (SS2). In the present study, we demonstrate that the gene encoding the SS2 precursor (PSS2) is actively expressed in the intermediate lobe of the frog pituitary. High concentrations of PSS2 mRNA have been detected by Northern blot analysis and in situ hybridization in the frog pars intermedia but not in the pars distalis or pars nervosa. The distribution of PSS1- and PSS2-derived peptides has been investigated by immunohistochemistry using two antisera directed against SS1 and the sequence 54-66 of PSS2 (PSS2(54-66)), respectively. The SS1 antiserum stained only a network of fibers in the neural lobe and a few nerve processes in the intermediate lobe. In contrast, the PSS2(54-66) antiserum produced intense labeling of melanotrope cells in the pars intermedia. Biochemical characterization of the immunoreactive materials present in pituitary extracts was performed by combining high-performance liquid chromatography analysis and RIA detection. The SS1 RIA revealed the existence of two major immunoreactive peaks that exhibited the same retention times as synthetic SS1 and SS2. The PSS2(54-66) RIA detected a single peak that likely corresponds to the N-flanking peptide of SS2 (PSS2(1-66)). The present study reveals that melanotrope cells of the frog pituitary selectively express the PSS2 gene and fully process PSS2 to generate the mature somatostatin variant SS2. Taken together, these data provide the first evidence that the gene encoding a hypophysiotropic neuropeptide is intensely expressed in the intermediate lobe of the pituitary.

Neuropeptide Y inhibits spontaneous alpha-melanocyte-stimulating hormone (alpha-MSH) release via a Y(5) receptor and suppresses thyrotropin-releasing hormone-induced alpha-MSH secretion via a Y(1) receptor in frog melanotrope cells.

In amphibians, the secretion of alpha-MSH by melanotrope cells is stimulated by TRH and inhibited by NPY. We have previously shown that NPY abrogates the stimulatory effect of TRH on alpha-MSH secretion. The aim of the present study was to characterize the receptor subtypes mediating the action of NPY and to investigate the intracellular mechanisms involved in the inhibitory effect of NPY on basal and TRH-induced alpha-MSH secretion. Y(1) and Y(5) receptor mRNAs were detected by RT-PCR and visualized by in situ hybridization histochemistry in the intermediate lobe of the pituitary. Various NPY analogs inhibited in a dose-dependent manner the spontaneous secretion of alpha-MSH from perifused frog neurointermediate lobes with the following order of potency porcine peptide YY (pPYY) > frog NPY (fNPY) > porcine NPY (pNPY)-2-36) > pNPY-(13-36) > [D-Trp(32)]pNPY > [Leu(31),Pro(34)]pNPY. The stimulatory effect of TRH (10(-8)6 M) on alpha-MSH release was inhibited by fNPY, pPYY, and [Leu(31),Pro(34)]pNPY, but not by pNPY-(13-36) and [D-Trp(32)]pNPY. These data indicate that the inhibitory effect of fNPY on spontaneous alpha-MSH release is preferentially mediated through Y(5) receptors, whereas the suppression of TRH-induced alpha-MSH secretion by fNPY probably involves Y(1) receptors. Pretreatment of neurointermediate lobes with pertussis toxin (PTX; 1 microg/ml; 12 h) did not abolish the inhibitory effect of fNPY on cAMP formation and spontaneous alpha-MSH release, but restored the stimulatory effect of TRH on alpha-MSH secretion, indicating that the adenylyl cyclase pathway is not involved in the action of fNPY on TRH-evoked alpha-MSH secretion. In the majority of melanotrope cells, TRH induces a sustained and biphasic increase in cytosolic Ca(2+) concentration. Preincubation of cultured cells with fNPY (10(-7) M) or omega-conotoxin GVIA (10(-7) M) suppressed the plateau phase of the Ca(2+) response induced by TRH. However, although fNPY abrogated TRH-evoked alpha-MSH secretion, omega-conotoxin did not, showing dissociation between the cytosolic Ca(2+) concentration increase and the secretory response. Collectively, these data indicate that in frog melanotrope cells NPY inhibits spontaneous alpha-MSH release and cAMP formation through activation of a Y(5) receptor coupled to PTX- insensitive G protein, whereas NPY suppresses the stimulatory effect of TRH on alpha-MSH secretion through a Y(1) receptor coupled to a PTX-sensitive G protein-coupled receptor.