An indispensable role of secretin in mediating the osmoregulatory functions of angiotensin II.

Fluid balance is critical to life and hence is tightly controlled in the body. Angiotensin II (ANGII), one of the most important components of this regulatory system, is recognized as a dipsogenic hormone that stimulates vasopressin (VP) expression and release. However, detailed mechanisms regarding how ANGII brings about these changes are not fully understood. In the present study, we show initially that the osmoregulatory functions of secretin (SCT) in the brain are similar to those of ANGII in mice and, more important, we discovered the role of SCT as the link between ANGII and its downstream effects. This was substantiated by the use of two knockout mice, SCTR(-/-) and SCT(-/-), in which we show the absence of an intact SCT/secretin receptor (SCTR) axis resulted in an abolishment or much reduced ANGII osmoregulatory functions. By immunohistochemical staining and in situ hybridization, the proteins and transcripts of SCT and its receptor are found in the paraventricular nucleus (PVN) and lamina terminalis. We propose that SCT produced in the circumventricular organs is transported and released in the PVN to stimulate vasopressin expression and release. In summary, our findings identify SCT and SCTR as novel elements of the ANGII osmoregulatory pathway in maintaining fluid balance in the body.

Multiple actions of secretin in the human body.

The discovery of secretin initiated the field of endocrinology. Over the past century, multiple gastrointestinal functions of secretin have been extensively studied, and it was discovered that the principal function of this peptide in the gastrointestinal system is to facilitate digestion and to provide protection. In view of the late identification of secretin and the secretin receptor in various tissues, including the central nervous system, the pleiotropic functions of secretin have more recently been an area of intense focus. Secretin is a classical hormone, and recent studies clearly showed secretin's involvement in neural and neuroendocrine pathways, although the neuroactivity and neural regulation of its release are yet to be elucidated. This chapter reviews our current understanding of the pleiotropic actions of secretin with a special focus on the hormonal and neural interdependent pathways that mediate these actions.

Southwestern blotting in investigating transcriptional regulation.

Southwestern blotting is used to investigate DNA-protein interactions. The advantage of this technique over other related methods such as electrophoretic mobility shift assay (EMSA) and DNA footprinting is that it provides information regarding the molecular weight of unknown protein factor. This method combines the features of Southern and Western blotting techniques; a denaturing SDS-PAGE is first employed to separate proteins electrophoretically based on size, and after transferring the proteins to a membrane support, the membrane-bound proteins are renatured and incubated with a (32)P-labeled double-stranded oligonucleotide probe of specific DNA sequence. The interaction of the probe with the protein(s) is later visualized by autoradiography. This technique could be combined with database searching (TransFac, http://www.gene-regulation.com/pub/databases.html#transfac), prediction of potential protein factors binding onto a target motif (e.g., Patch search), in vitro supershift EMSA and in vivo chromatin immunoprecipitation (ChIP) assays for effective identification of protein factors. The whole Southwestern blotting procedure takes approximately 4 d to complete. In this article, a commonly used protocol and expected results are described and discussed.

Discovery of growth hormone-releasing hormones and receptors in nonmammalian vertebrates.

In mammals, growth hormone-releasing hormone (GHRH) is the most important neuroendocrine factor that stimulates the release of growth hormone (GH) from the anterior pituitary. In nonmammalian vertebrates, however, the previously named GHRH-like peptides were unable to demonstrate robust GH-releasing activities. In this article, we provide evidence that these GHRH-like peptides are homologues of mammalian PACAP-related peptides (PRP). Instead, GHRH peptides encoded in cDNAs isolated from goldfish, zebrafish, and African clawed frog were identified. Moreover, receptors specific for these GHRHs were characterized from goldfish and zebrafish. These GHRHs and GHRH receptors (GHRH-Rs) are phylogenetically and structurally more similar to their mammalian counterparts than the previously named GHRH-like peptides and GHRH-like receptors. Information regarding their chromosomal locations and organization of neighboring genes confirmed that they share the same origins as the mammalian genes. Functionally, the goldfish GHRH dose-dependently activates cAMP production in receptor-transfected CHO cells as well as GH release from goldfish pituitary cells. Tissue distribution studies showed that the goldfish GHRH is expressed almost exclusively in the brain, whereas the goldfish GHRH-R is actively expressed in brain and pituitary. Taken together, these results provide evidence for a previously uncharacterized GHRH-GHRH-R axis in nonmammalian vertebrates. Based on these data, a comprehensive evolutionary scheme for GHRH, PRP-PACAP, and PHI-VIP genes in relation to three rounds of genome duplication early on in vertebrate evolution is proposed. These GHRHs, also found in flounder, Fugu, medaka, stickleback, Tetraodon, and rainbow trout, provide research directions regarding the neuroendocrine control of growth in vertebrates.

The prenatal expression of secretin receptor.

Secretin is a classical gastrointestinal peptide while its neuroactive functions in the central nervous system have recently been consolidated. In the past, there was little information regarding the expression of secretin receptor in prenatal development. In this article, using mouse embryos and by in situ hybridization, secretin receptor transcripts were detected in several developing brain regions including the cerebellar primordium and choroid plexus. In the developing intestine, secretin receptor is present in the epithelial lining of the villi and the inner circular muscle. Interestingly, the transcripts for secretin receptor were also detected in the epicardium and myocardium of the developing heart as well as the glomerulus and collecting duct in the developing kidney. Taken together, our data suggest a potential pleiotrophic role of secretin during embryonic development.

Signaling mechanisms of secretin receptor.

Secretin, a 27-amino acid gastrointestinal peptide, was initially discovered based on its activities in stimulating pancreatic juice. In the past 20 years, secretin was demonstrated to exhibit pleiotropic functions in many different tissues and more importantly, its role as a neuropeptide was substantiated. To carry out its activities in the central nervous system and in peripheral organs, secretin interacts specifically with one known receptor. Secretin receptor, a member of guanine nucleotide-binding protein (G protein)-coupled receptor (GPCR) in the secretin/VIP/glucagon subfamily, possesses the characteristics of GPCR with seven conserved transmembrane domains, a relatively large amino-terminal extracellular domain and an intracellular carboxyl terminus. The structural features and signal transduction pathways of the secretin receptor in various tissues are reviewed in this article.

Secretin, a known gastrointestinal peptide, is widely expressed during mouse embryonic development.

The gastrointestinal functions of the 27-amino acid secretin peptide have been well established. In previous prenatal studies, secretin expression in the rat duodenum was reported after day 17 of gestation while its expression in other organs and its functions in the developing embryos are still unknown. By in situ hybridization and immunohistochemical staining, secretin transcripts and peptides were found to be widely expressed in mouse embryos. Consistent with the idea that secretin is a brain-gut peptide, its expressions are present in several developing brain regions such as cephalic mesenchyme, cerebellar primordium and choroid plexus as well as the epithelial villi lining and inner circular muscle of the developing intestine. Other than these organs, secretin was also detected in the developing heart including the ventricular epicardium and myocardium and certain structures of the developing kidney like ureteric bud, collecting duct and glomerulus. These observations strongly suggest for a functional role of secretin during mouse embryonic development.