Cloning of two members of the calcitonin-family receptors from stingray, Dasyatis akajei: possible physiological roles of the calcitonin family in osmoregulation

In cartilaginous fish, two cDNAs encoding calcitonin-family receptors were isolated for the first time from the stingray brain. The open reading frame of one receptor cDNA coded a 525-amino acid protein. The amino acid identity of this receptor to human calcitonin-receptor-like receptor (CRLR) is 64.5%, frog CRLR is 64.7%, and flounder CRLR is 61.2% and this was higher than to human calcitonin receptor (CTR) (46.1%), frog CTR (54.7%), and flounder CTR (48.9%). We strongly suggested that this receptor is a ray CRLR based on phylogenetic analysis. In case of the second receptor, amino acid identity among CRLRs (human 50.5%, frog 50.7%, flounder 48.0%) and CTRs (human 43.2%, frog 49.1%, flounder 41.8%) was similar. From phylogenetic analysis of both CRLRs and CTRs, we believe that this receptor is ray CTR. The expression of ray CRLR mRNA was predominantly detected in the nervous system (brain) and vascular system (atrium, ventricle, and gill), which reflects the similar localization of CGRP in the nervous and vascular systems as mammals. It was observed that the second receptor was expressed in several tissues, namely cartilage, brain, pituitary gland, gill, atrium, ventricle, pancreas, spleen, liver, gall bladder, intestine, rectal gland, kidney, testis and ovary. This localization pattern was very similar to flounder CTR. Both receptor mRNAs were strongly expressed in the gill. This suggests that the calcitonin-family members are involved in the osmoregulation of stingray as this fish is known to be euryhaline. When a stingray was transferred to diluted seawater (20% seawater), the expression of both receptors significantly decreased in the gill. Similar results were obtained in the kidney of the stingray. Thus, our cloning and isolation of both receptors in the stingray will be helpful for elucidation of their physiological role(s) such as osmoregulation including calcium metabolism of cartilaginous fish.     

Altered expression of neprilysin family members in the pituitary gland of sleep-disturbed rats, an animal model of severe fatigue

Alterations of the expression of some peptidases in the pituitary gland of a fatigued rat model were identified. Rats were kept in a cage filled with water to a height of 1.5 cm to disturb deep sleep. After 24-h sleep disturbance, expression of neutral endopeptidase 24.11 (neprilysin) mRNA was increased in the intermediate lobe of the pituitary gland, whereas the mRNA expression of another family member, damage-induced neuronal endopeptidase, which is normally expressed in a subgroup of anterior pituitary cells, was significantly suppressed. These alterations were demonstrated by RT-PCR, northern blotting and in situ hybridization. Other family members, such as neprilysin 2 and endothelin converting enzyme-1, did not show any change in mRNA expression. An increase of neprilysin mRNA expression was not seen in any other tissues of the sleep-disturbed rats. The enzymatic activity of neprilysin was also increased in the pituitary. The augmentation of neprilysin expression and activity was prolonged as long as the sleep disturbance continued (up to 5 days), and returned to the basal level when rats were allowed to sleep freely. These results suggest that peptide processing and degradation in the pituitary may be an influential factor in fatigued states such as sleep disturbance.     

Differential gene expression and regulation of type-1 angiotensin II receptor subtypes in the rat.

A simplified and sensitive method for measuring expression levels of type-1 angiotensin II (AT1) receptor subtypes, AT1A and AT1B, was established. The two receptor cDNAs were co-amplified and measured by polymerase chain reaction using primers based on the corresponding receptor subtype genes. Both AT1A and AT1B mRNAs were widely expressed in the rat tissues including adrenal gland, kidney, heart, aorta, lung, liver, testis, pituitary gland, cerebrum and cerebellum. AT1A mRNA was predominantly expressed in the rat tissues examined except adrenal gland and pituitary gland where AT1B mRNA was predominantly expressed. Sodium depletion did not change mRNA levels of AT1A and AT1B in the all tissues. However, both AT1A and AT1B mRNA levels in the heart and aorta were down-regulated by treatment with AT1 specific antagonist, TCV 116. In contrast, AT1B mRNA in the adrenal gland was mainly reduced by the treatment. These results suggest that the expression level of AT1B mRNA in the adrenal gland depends on the activity of the renin-angiotensin-aldosterone system (RAAS) and both receptor subtypes mediate contraction and hypertrophy of the smooth and cardiac muscles via the RAAS.     

Expression of orexin receptors in the brain and peripheral tissues of the male sheep

Orexins exert their effects through two specific receptors (OX1R and OX2R) that have been found mainly in the brain and also in peripheral tissues of rats and humans. Here, we demonstrate expression of mRNA encoding for ovine OX1R and OX2R in central and peripheral tissues of sheep. Gene expression for orexin receptors in the hypothalamus and the preoptic area was localised by in situ hybridisation. OX1R was detected in arcuate nuclei (ARC), median eminence (ME), the lateral hypothalamic nuclei and preoptic area (POA) and it was scattered along the third ventricle from the paraventricular (PVN) to the ventromedial hypothalamic nuclei (VMH). OX2R was localised in the PVN, ARC, ME, ventral VMH and a small region of the ventral POA. Gene expression for OX1R and OX2R in central and peripheral tissues was analysed using quantitative real time RT-PCR. Both orexin receptor genes were expressed in the hypothalamus, POA, hippocampus, amygdala, olfactory bulb, pineal gland and recess and pituitary gland, whereas only OX1R mRNA was detected in the testis, kidney and adrenal gland. The expression of the genes for orexin receptors in this range of ovine tissues suggests roles for orexins in multiple physiological functions, with actions at both central and peripheral levels.     

Hypophyseal corticosteroids stimulate somatotrope differentiation in the embryonic chicken pituitary gland

Although it is known that glucocorticoids induce differentiation of growth hormone (GH)-producing cells in rodents and birds, the effect of mineralocorticoids on GH mRNA expression and the origin of corticosteroids affecting somatotrope differentiation have not been elucidated. In this study, we therefore carried out experiments to determine the effect of mineralocorticoids on GH mRNA expression in the chicken anterior pituitary gland in vitro and to determine whether corticosteroids are synthesized in the chicken embryonic pituitary gland. In a pituitary culture experiment with E11 embryos, both corticosterone and aldosterone stimulated GH mRNA expression and increased the number of GH cells in both lobes of the pituitary gland in a dose-dependent manner. These effects of the corticosteroids were significantly reversed by pretreatment with mifepristone, a glucocorticoid receptor (GR) antagonist, or spironolactone, a mineralocorticoid receptor (MR) antagonist. Interestingly, an in vitro serum-free culture experiment with an E11 pituitary gland showed that the GH mRNA level spontaneously increased during cultivation for 2 days without any extra stimulation, and this increase in GH mRNA level was completely suppressed by metyrapone, a corticosterone-producing enzyme P450C11 inhibitor. Moreover, progesterone, the corticosterone precursor, also stimulated GH mRNA expression in the cultured chicken pituitary gland, and this effect was blocked by pretreatment with metyrapone. We also detected mRNA expression of enzymes of cytochrome P450 cholesterol side chain cleavage (P450scc) and 3β-hydroxysteroid dehydrogenase1 (3β-HSD1) in the developmental chicken pituitary gland from E14 and E18, respectively. These results suggest that mineralocorticoids as well as glucocorticoids can stimulate GH mRNA expression and that corticosteroids generated in the embryonic pituitary gland by intrinsic steroidogenic enzymes stimulate somatotrope differentiation.     

Molecular cloning and expression of a pituitary-specific receptor for growth hormone-releasing hormone.

A novel cDNA was isolated from rat pituitary mRNA using the polymerase chain reaction to amplify sequences encoding G protein-coupled receptors. The human homolog of this cDNA was isolated and expressed in human kidney 293 cells, and membrane fractions from these cells were found to bind human GH-releasing hormone (GHRH) with high affinity and specificity. GHRH also stimulates intracellular cAMP production in these transfected cells. The encoded receptor protein contains seven potential membrane-spanning domains, a hallmark of G protein-coupled receptors, and is homologous to previously identified receptors for secretin and vasoactive intestinal peptide, ligands that are related to GHRH. The rat GHRH receptor mRNA is expressed predominantly, if not exclusively, in the anterior pituitary gland, the major target for GHRH action. These results define a mechanism for cellular signaling by GHRH and provide the opportunity to examine the role of the GHRH receptor in growth abnormalities that involve the GH axis.     

Characterization of two duplicate zebrafish Cb2-like cannabinoid receptors

Several cannabinoid receptors have been detected in many organisms. The best known are CB1, mainly expressed in the central nervous system and CB2 which is almost exclusively expressed in the periphery. Here we report the molecular characterization of two duplicate CB2-like cannabinoid receptors from zebrafish (Danio rerio) (zebrafish Cb2a and zebrafish Cb2b). The amino acid sequences of these receptors present 56% identity with Takifugu rubripes CB2 sequence and 39% with human CB2 sequence and conserve some specific key residues for cannabinoid receptor function. Both duplicate receptors are expressed in peripheral tissues (gills, heart, intestine and muscle), immune tissue (spleen) and also in the central nervous system. Using in situ hybridization techniques zebrafish Cb2 mRNA expression was observed for the first time in the adenohypophysial cells of the rostral pars distalis and proximal pars distalis of the pituitary gland. Given the importance of the existence of duplication of genes in teleosts, the combined analysis of these two new cannabinoid receptors opens a new exciting door to investigate and understand cannabinoid function throughout evolution.     

Retinoic acid stimulates growth hormone synthesis in human somatotrophic adenoma cells: Characterization of its nuclear receptors

In order to gain a better understanding on the possible role of retinoic acid (RA) on human GH secretion, we have characterized the expression of its nuclear receptors in somatotropic adenoma cell extracts. By immunoblotting with rabbit polyclonal antibodies directed against RARα, β, and γ and RXRα and β, we could only detect the presence of RARα and RXRα proteins. The predominant expression of RXRα was confirmed at the mRNA level by Northern and slot-blot analysis. When then investigated the effect of RA on GH synthesis in cell culture of adenomatous somatotrophs. In cultured cells, RA (1 μM) stimulated GH secretion, increased intracellular GH content and GH mRNA levels within 72 h, suggesting a modulation of GH synthesis by RA. J. Cell. Biochem 65:25–31. © 1997 Wiley-Liss, Inc.     

Expression of retinaldehyde dehydrogenase 1 in the anterior pituitary glands of adult rats

Retinoic acid (RA) plays a critical role in cell growth and tissue development and is also a regulatory factor of pituitary function. However, whether RA is generated in the pituitary gland and plays a role as a paracrine and/or autocrine factor is generally unknown. RA is synthesized from retinoids through oxidation processes. Dehydrogenases that catalyze the oxidation of retinal to RA are members of the retinaldehyde dehydrogenase (RALDH) family. Recently, we demonstrated that RALDH2 and RALDH3, but not RALDH1, were expressed in the developing anterior pituitary gland of rats, but the expression of RALDHs in the adult pituitary gland was not determined. Therefore, we have now examined the expression of RALDH1, RALDH2, and RALDH3 mRNAs in the pituitary gland of adult rats. Analysis by quantitative real-time polymerase chain reaction of adult pituitary glands has revealed a high level of RALDH1 mRNA but not of RALDH2 mRNA or RALDH3 mRNA. We have also detected mRNA expression for RALDH1 in the anterior pituitary gland by in situ hybridization with digoxigenin-labeled cRNA probes. Double-staining for RALDH1 mRNA and pituitary hormones or S-100 protein, a marker of folliculo-stellate cells (FS-cells), has revealed RALDH1 mRNA expression in a portion of prolactin-producing cells, marginal layer cells, and FS-cells. Our results suggest that RA is generated in the adult anterior pituitary gland, and that it may act locally on pituitary cells. This work was supported by a Grant-in-Aid for Young Scientists (B) from the Japanese Ministry of Education, Culture, Sports, Science, and Technology (18790149) and by the Foundation of Growth Science.     

Early expression of thyroid hormone receptor beta and retinoid X receptor gamma in the Xenopus embryo

The role of thyroid hormone in Xenopus metamorphosis is particularly well understood as it plays an essential role in that process. However, recent evidence suggests that thyroid hormone may play an earlier role in amphibian embryogenesis. We demonstrate that Xenopus thyroid hormone receptor beta (XTR beta) is expressed shortly after neural fold closure, and that its expression is localized to the developing retina. Retinoid X receptor gamma (RXR gamma), a potential dimerization partner for XTR beta, was also found to be expressed in the retina at early stages, and at later stages RXR gamma was also expressed in the liver diverticulum. Addition of either thyroid hormone or 9-cis retinoic acid, the ligands for XTR beta and RXR gamma, respectively, did not alter the expression of their receptors. However, the addition of thyroid hormone and 9-cis retinoic acid did alter rhodopsin mRNA expression. Addition of thyroid hormone generates a small expansion of the rhodopsin expression domain. When 9-cis retinoic acid or a combination of thyroid hormone and 9-cis retinoic acid was administered, there was a decrease in the expression domain of rhodopsin in the developing retina. These results provide evidence for an early role for XTR beta and RXR gamma in the developing Xenopus retina.     

Induction of Src-suppressed C kinase substrate (SSeCKS) in vascular endothelial cells by bacterial lipopolysaccharide.

We isolated cDNA of the mouse homologue of the src-suppressed C kinase substrate (SSeCKS) and analyzed the effects of lipopolysaccharide (LPS) injection on the tissue expression pattern of this protein. Northern blotting analysis showed that SSeCKS mRNA was expressed abundantly in the testis but at undetectable levels in other tissues of untreated control mice. Intraperitoneal administration of LPS strongly induced SSeCKS mRNA expression in the lung, heart, liver, spleen, kidney, lymph node, adrenal gland, and pituitary gland, as well as in the brain. In lung and spleen, the SSeCKS mRNA levels increased almost 10-fold at 1 hr after LPS injection and persisted at high levels until 4 hr. Both in situ hybridization and immunohistochemical studies revealed that LPS administration conspicuously elevated expression of SSeCKS mRNA and protein in vascular endothelial cells of several organs. Ectopic expression of SSeCKS caused loss of cytoplasmic F-actin fibers in the mouse endothelial cell line LEII. These results indicate that SSeCKS is one of the major LPS-responsive proteins and may participate in alteration of cytoskeletal architecture in endothelial cells during inflammation.