Loss of the Secretin Receptor Impairs Renal Bicarbonate Excretion and Aggravates Metabolic Alkalosis in Mice during Acute Base-Loading.

SIGNIFICANCE STATEMENT: During acute base excess, the renal collecting duct ß -intercalated cells ( ß -ICs) become activated to increase urine base excretion. This process is dependent on pendrin and cystic fibrosis transmembrane regulator (CFTR) expressed in the apical membrane of ß -ICs. The signal that leads to activation of this process was unknown. Plasma secretin levels increase during acute alkalosis, and the secretin receptor (SCTR) is functionally expressed in ß -ICs. We find that mice with global knockout for the SCTR lose their ability to acutely increase renal base excretion. This forces the mice to lower their ventilation to cope with this challenge. Our findings suggest that secretin is a systemic bicarbonate-regulating hormone, likely being released from the small intestine during alkalosis. BACKGROUND: The secretin receptor (SCTR) is functionally expressed in the basolateral membrane of the ß -intercalated cells of the kidney cortical collecting duct and stimulates urine alkalization by activating the ß -intercalated cells. Interestingly, the plasma secretin level increases during acute metabolic alkalosis, but its role in systemic acid-base homeostasis was unclear. We hypothesized that the SCTR system is essential for renal base excretion during acute metabolic alkalosis. METHODS: We conducted bladder catheterization experiments, metabolic cage studies, blood gas analysis, barometric respirometry, perfusion of isolated cortical collecting ducts, immunoblotting, and immunohistochemistry in SCTR wild-type and knockout (KO) mice. We also perfused isolated rat small intestines to study secretin release. RESULTS: In wild-type mice, secretin acutely increased urine pH and pendrin function in isolated perfused cortical collecting ducts. These effects were absent in KO mice, which also did not sufficiently increase renal base excretion during acute base loading. In line with these findings, KO mice developed prolonged metabolic alkalosis when exposed to acute oral or intraperitoneal base loading. Furthermore, KO mice exhibited transient but marked hypoventilation after acute base loading. In rats, increased blood alkalinity of the perfused upper small intestine increased venous secretin release. CONCLUSIONS: Our results suggest that loss of SCTR impairs the appropriate increase of renal base excretion during acute base loading and that SCTR is necessary for acute correction of metabolic alkalosis. In addition, our findings suggest that blood alkalinity increases secretin release from the small intestine and that secretin action is critical for bicarbonate homeostasis.

Novel small molecule MRGPRX2 antagonists inhibit a murine model of allergic reaction.

BACKGROUND: Activation of Mas-related G protein-coupled receptor X2 (MRGPRX2) is a crucial non-IgE pathway for mast cell activation associated with allergic reactions and inflammation. Only a few peptides and small compounds targeting MRGPRX2 have been reported, with limited information on their pharmacologic activity. OBJECTIVE: We sought to develop novel small molecule MRGPRX2 antagonists to treat MRGPRX2-mediated allergies and inflammation. METHODS: A computational approach was used to design novel small molecules as MRGPRX2 antagonists. The short-listed molecules were synthesized and characterized by liquid chromatography and mass spectrometry as well as nuclear magnetic resonance. Inhibitory activity on MRGPRX2 signaling was assessed in vitro by using functional bioassays (ß-hexosaminidase, calcium flux, and chemokine synthesis) and receptor activation assays (ß-arrestin recruitment and Western blot analysis) in human LAD-2 mast cells and HTLA cells. In vivo effects of the novel MRGPRX2 antagonists were assessed using a mouse model of acute allergy and systemic anaphylaxis. RESULTS: The novel small molecules demonstrated higher binding affinity with MRGPRX2 in the docking study. The half-maximal inhibitory concentration is in the low micromolar range (5-21 µM). The small molecules inhibited not only the early phase of mast cell activation but also the late phase, associated with chemokine and prostaglandin release. Further, Western blot analysis revealed inhibition of downstream phospholipase C-γ, extracellular signal-regulated protein kinase 1/2, and Akt signaling pathway. Moreover, in the mouse models of allergies, small molecule administration effectively blocks acute, systemic allergic reactions and inflammation and prevents systemic anaphylaxis. CONCLUSION: The small molecules might hold a significant therapeutic promise to treat MRGPRX2-mediated allergies and inflammation.

P17 induces chemotaxis and differentiation of monocytes via MRGPRX2-mediated mast cell-line activation.

BACKGROUND: P17, a peptide isolated from Tetramorium bicarinatum ant venom, is known to induce an alternative phenotype of human monocyte-derived macrophages via activation of an unknown G protein-coupled receptor (GPCR). OBJECTIVE: We sought to investigate the mechanism of action and the immunomodulatory effects of P17 mediated through MRGPRX2 (Mas-related G protein-coupled receptor X2). METHODS: To identify the GPCR for P17, we screened 314 GPCRs. Upon identification of MRGPRX2, a battery of in silico, in vitro, ex vivo, and in vivo assays along with the receptor mutation studies were performed. In particular, to investigate the immunomodulatory actions, we used ß-hexosaminidase release assay, cytokine releases, quantification of mRNA expression, cell migration and differentiation assays, immunohistochemical labeling, hematoxylin and eosin, and immunofluorescence staining. RESULTS: P17 activated MRGPRX2 in a dose-dependent manner in ß-arrestin recruitment assay. In LAD2 cells, P17 induced calcium and ß-hexosaminidase release. Quercetin- and short hairpin RNA-mediated knockdown of MRGPRX2 reduced P17-evoked ß-hexosaminidase release. In silico and in vitro mutagenesis studies showed that residue Lys8 of P17 formed a cation-π interaction with the Phe172 of MRGPRX2 and [Ala8]P17 lost its activity partially. P17 activated LAD2 cells to recruit THP-1 and human monocytes in Transwell migration assay, whereas MRGPRX2-impaired LAD2 cells cannot. In addition, P17-treated LAD2 cells stimulated differentiation of THP-1 and human monocytes, as indicated by the enhanced expression of macrophage markers cluster of differentiation 11b and TNF-α by quantitative RT-PCR. Immunohistochemical and immunofluorescent staining suggested monocyte recruitment in mice ears injected with P17. CONCLUSIONS: Our data provide novel structural information regarding the interaction of P17 with MRGPRX2 and intracellular pathways for its immunomodulatory action.

GIP receptor suppresses PAC1receptor-mediated neuronal differentiation via formation of a receptor heterocomplex.

Pituitary adenylate cyclase-activating peptide (PACAP) receptor (PAC1R) is a class B Gprotein-coupled receptor (GPCR) that is widely expressed in the human body and is involved in neuronal differentiation. As class B GPCRs are known to form heterocomplexes with family members, we hypothesized that PAC1R mediates neuronal differentiation through interaction with a class B GPCR. We used the BRET assay to identify potential interactions between PAC1R and 11 class B GPCRs. Gastric inhibitory polypeptide receptor (GIPR) and secretin receptor were identified as putative binding partners of PAC1R. The effect of heterocomplex formation by PAC1R on receptor activation was evaluated with the cyclic (c)AMP, luciferase reporter, and calcium signaling assays; and the effects on receptor internalization and subcellular localization were examined by confocal microscopy. The results suggested he PAC1R/GIPR heterocomplex suppressed signaling events downstream of PAC1R, including cAMP production, serum response element and calcium signaling, and ß-arrestin recruitment. Protein-protein interaction was analyzed in silico, and induction of neuronal differentiation by the PAC1R heterocomplex was assessed in SH-SY5Y neuronal cells by measure the morphological changes and marker genes expression by real-time quantitative PCR and western blot. Over-expression of GIPR suppressed PACAP/PAC1R-mediated neuronal differentiation and the differentiation markers expression in SH-SY5Y cells. GIPR regulates neuronal differentiation through heterocomplex formation with PAC1R.

A crustacean annotated transcriptome (CAT) database.

BACKGROUND: Decapods are an order of crustaceans which includes shrimps, crabs, lobsters and crayfish. They occur worldwide and are of great scientific interest as well as being of ecological and economic importance in fisheries and aquaculture. However, our knowledge of their biology mainly comes from the group which is most closely related to crustaceans - insects. Here we produce a de novo transcriptome database, crustacean annotated transcriptome (CAT) database, spanning multiple tissues and the life stages of seven crustaceans. DESCRIPTION: A total of 71 transcriptome assemblies from six decapod species and a stomatopod species, including the coral shrimp Stenopus hispidus, the cherry shrimp Neocaridina davidi, the redclaw crayfish Cherax quadricarinatus, the spiny lobster Panulirus ornatus, the red king crab Paralithodes camtschaticus, the coconut crab Birgus latro, and the zebra mantis shrimp Lysiosquillina maculata, were generated. Differential gene expression analyses within species were generated as a reference and included in a graphical user interface database at http://cat.sls.cuhk.edu.hk/. Users can carry out gene name searches and also access gene sequences based on a sequence query using the BLAST search function. CONCLUSIONS: The data generated and deposited in this database offers a valuable resource for the further study of these crustaceans, as well as being of use in aquaculture development.

In vivo actions of SCTR/AT1aR heteromer in controlling Vp expression and release via cFos/cAMP/CREB pathway in magnocellular neurons of PVN.

With an increasing body of evidence regarding GPCR oligomerization and its clinical implications over the last decade, the modulation and dynamics of GPCR homo- and hetero-oligomers has more recently become an area of intense research focus. Previously, our lab showed in vitro heteromer formation between angiotensin II receptor type 1 subtype a (AT1aR) and secretin receptor (SCTR), which is involved in in vivo control of hyperosmolality-induced water drinking behavior. Because the secretin (SCT)/SCTR axis is crucial to the central actions of angiotensin II (ANGII) and both SCT and ANGII are capable of triggering vasopressin (Vp) release from hypothalamus, we investigated here the in vivo role of SCTR-AT1aR heteromer in regulating Vp release in hypothalamus using transmembrane peptides as tools. We showed that SCTR-AT1aR heteromer mediates stimulatory actions of both SCT and ANGII in hypothalamic Vp expression and release as well as neuronal activities via the immediate early gene cFos. The results from this study not only are consistent with our hypothesis that SCT and ANGII interact at the receptor level to mediate their water homeostatic activities but also provide evidence for in vivo functions of cross-class GPCR heteromers.-Mak, S. O. K., Zhang, L., Chow, B. K. C. In vivo actions of SCTR/AT1aR heteromer in controlling Vp expression and release via cFos/cAMP/CREB pathway in magnocellular neurons of PVN.

Secretin is involved in sodium conservation through the renin-angiotensin-aldosterone system.

Secretin (SCT) and its receptor (SCTR) are important in fluid regulation at multiple levels via the modulation of expression and translocation of renal aquaporin 2 and functions of central angiotensin II (ANGII). The functional interaction of SCT with peripheral ANGII, however, remains unknown. As the ANGII-aldosterone axis dominates the regulation of renal epithelial sodium channel (ENaC) function, we therefore tested whether SCT/SCTR can regulate sodium homeostasis via the renin-angiotensin-aldosterone system. SCTR-knockout (SCTR-/-) mice showed impaired aldosterone synthase (CYP11B2) expression and, consequently, aldosterone release upon intraperitoneal injection of ANGII. Endogenous ANGII production induced by dietary sodium restriction was higher in SCTR-/- than in C57BL/6N [wild-type (WT)] mice, but CYP11B2 and aldosterone synthesis were not elevated. Reduced accumulation of cholesteryl ester-the precursor of aldosterone-was observed in adrenal glands of SCTR-/- mice that were fed a low-sodium diet. Absence of SCTR resulted in elevated basal transcript levels of adrenal CYP11B2 and renal ENaCs. Although transcript and protein levels of ENaCs were similar in WT and SCTR-/- mice under sodium restriction, ENaCs in SCTR-/- mice were less sensitive to amiloride hydrochloride. In summary, the SCT/SCTR axis is involved in aldosterone precursor uptake, and the knockout of SCTR results in defective aldosterone biosynthesis/release and altered sensitivity of ENaCs to amiloride.-Bai, J., Chow, B. K. C. Secretin is involved in sodium conservation through the renin-angiotensin-aldosterone system.

Structure and Function of Cross-class Complexes of G Protein-coupled Secretin and Angiotensin 1a Receptors.

Complexes of secretin (SecR) and angiotensin 1a (Atr1a) receptors have been proposed to be functionally important in osmoregulation, providing an explanation for overlapping and interdependent functions of hormones that bind and activate different classes of GPCRs. However, the nature of these cross-class complexes has not been well characterized and their signaling properties have not been systematically explored. We now use competitive inhibition of receptor bioluminescence resonance energy transfer and bimolecular fluorescence complementation to establish the dominant functionally important state as a symmetrical homodimeric form of SecR decorated by monomeric Atr1a, interacting through lipid-exposed faces of Atr1a TM1 and TM4. Conditions increasing prevalence of this complex exhibited negative allosteric modulatory impact on secretin-stimulated cAMP responses at SecR. In contrast, activating Atr1a with full agonist in such a complex exhibited a positive allosteric modulatory impact on the same signaling event. This modulation was functionally biased, with secretin-stimulated calcium responses unaffected, whereas angiotensin-stimulated calcium responses through the complex were reduced or absent. Further supporting this interpretation, Atr1a with mutations of lipid-exposed faces of TM1 and TM4 that did not affect its ability to bind or signal, could be expressed in the same cell as SecR, yet not exhibit either the negative or positive allosteric impact on cAMP observed with the inactive or activated states of wild type Atr1a on function, and not interfere with angiotensin-stimulated calcium responses like complexes with Atr1a. This may provide a more selective means of exploring the physiologic functional impact of this cross-class receptor complex without interfering with the function of either component receptor.

Role of nuclear factor of activated T-cells 5 in regulating hypertonic-mediated secretin receptor expression in kidney collecting duct cells.

A growing body of evidence suggests that secretin (SCT) is an important element in the osmoregulatory pathway. It is interesting to note that both SCT and its receptor (SCTR) gene are activated upon hyperosmolality in the kidney. However, the precise molecular mechanisms underlying the induction of the SCTR gene expression in response to changes in osmolality have yet to be clarified. Detailed DNA sequence analysis of the promoter regions of the SCTR gene reveals the presence of multiple osmotic response elements (ORE). The ORE is the binding site of a key osmosensitive transactivator, namely, the nuclear factor of activated T-cells 5 (NFAT5). SCTR and NFAT5 are co-expressed in the kidney cortex and medulla collecting duct cells. We therefore hypothesize that NFAT5 is responsible for modulating SCTR expression in hypertonic environments. In this study, we found hypertonicity stimulates the promoter activities and endogenous gene expression of SCTR in mouse kidney cortex collecting duct cells (M1) and inner medulla collecting duct cells (mIMCD3). The overexpression and silencing of NFAT5 further confirmed it to be responsible for the up-regulation of the SCTR gene under hypertonic conditions. A significant increase in the interaction between NFAT5 and the SCTR promoter was also observed following chromatin immunoprecipitation assay. In vivo, osmotic stress up-regulates the SCTR gene in the kidney cortex and medulla of wild-type mice, but does not do so in NFAT5(+/-) animals. Hence, this study provides comprehensive information on how NFAT5 regulates SCTR expression in different osmotic environments.

Secretin, at the hub of water-salt homeostasis.

Water and salt metabolism are tightly regulated processes. Maintaining this milieu intérieur within narrow limits is critical for normal physiological processes to take place. Disturbances to this balance can result in disease and even death. Some of the better-characterized regulators of water and salt homeostasis include angiotensin II, aldosterone, arginine vasopressin, and oxytocin. Although secretin (SCT) was first described >100 years ago, little is known about the role of this classic gastrointestinal hormone in the maintenance of water-salt homeostasis. In recent years, increasing body of evidence suggested that SCT and its receptor play important roles in the central nervous system and kidney to ensure that the mammalian extracellular fluid osmolarity is kept within a healthy range. In this review, we focus on recent advances in our understanding of the molecular, cellular, and network mechanisms by which SCT and its receptor mediate the control of osmotic homeostasis. Implications of hormonal cross talk and receptor-receptor interaction are highlighted.

PAN substitutions A37S, A37S/I61T and A37S/V63I attenuate the replication of H7N7 influenza A virus by impairing the polymerase and endonuclease activities.

Substitutions in the PA N-terminus (PAN) of influenza A viruses are associated with viral pathogenicity. During our previous study, which identified PAN-V63I and -A37S/I61T/V63I/V100A substitutions as virulence determinants, we observed a severe decrease in virus growth and transcription/replication capacity posed by PAN-A37S/V100A substitution. To further delineate the significance of substitutions at these positions, we generated mutant H7N7 viruses bearing the substitutions PAN-A37S, -A37S/I61T, -A37S/V63I, -V100A, -I61T/V100A and -V63I/V100A by reverse genetics. Our results showed that all mutant viruses except PAN-V100A showed a significantly reduced growth capability in infected cells. At the same time, the PAN-A37S, -A37S/I61T and -A37S/V63I mutant viruses displayed decreased viral transcription and replication by diminishing virus RNA synthesis activity. Biochemical assays indicated that the substitutions PAN-A37S, -A37S/I61T and -A37S/V63I suppressed the polymerase and endonuclease activities when compared with those of the wild-type. Together, our results demonstrated that the PAN-A37S, -A37S/I61T and -A37S/V63I substitutions contributed to a decreased pathogenicity of avian H7N7 influenza A virus.

Functional Pairing of Class B1 Ligand-GPCR in Cephalochordate Provides Evidence of the Origin of PTH and PACAP/Glucagon Receptor Family.

Several hypotheses have been proposed regarding the origin and evolution of the secretin family of peptides and receptors. However, identification of homologous ligand-receptor pairs in invertebrates and vertebrates is difficult because of the low levels of sequence identity between orthologs of distant species. In this study, five receptors structurally related to the vertebrate class B1 G protein-coupled receptor (GPCR) family were characterized from amphioxus (Branchiostoma floridae). Phylogenetic analysis showed that they clustered with vertebrate parathyroid hormone receptors (PTHR) and pituitary adenylate cyclase-activating polypeptide (PACAP)/glucagon receptors. These PTHR-like receptors shared synteny with several PTH and PACAP/glucagon receptors identified in spotted gar, Xenopus, and human, indicating that amphioxus preserves the ancestral chordate genomic organization of these receptor subfamilies. According to recent data by Mirabeau and Joly, amphioxus also expresses putative peptide ligands including homologs of PTH (bfPTH1 and 2) and PACAP/GLUC-like peptides (bfPACAP/GLUCs) that may interact with these receptors. Functional analyses showed that bfPTH1 and bfPTH2 activated one of the amphioxus receptors (bf98C) whereas bfPACAP/GLUCs strongly interacted with bf95. In summary, our data confirm the presence of PTH and PACAP/GLUC ligand-receptor pairs in amphioxus, demonstrating that functional homologs of vertebrate PTH and PACAP/glucagon GPCR subfamilies arose before the cephalochordate divergence from the ancestor of tunicates and vertebrates.

A novel small-molecule compound disrupts influenza A virus PB2 cap-binding and inhibits viral replication.

OBJECTIVES: The conserved residues 318-483 in the PB2 subunit of influenza A polymerase is an independently folded cap-binding domain (PB2cap) that exhibits a distinct binding mode from other host cap-binding proteins, which suggests that PB2cap might be an ideal drug target. This study aimed to identify a new class of anti-influenza inhibitors that specifically disrupts the interaction between PB2cap and host cap structures. METHODS: An innovative fluorescence polarization assay was established for primary screening, followed by cap-binding inhibitory activity, antiviral efficacy and cytotoxicity evaluations of the selected compounds. The best compound was characterized by multi-cycle virus growth assay, cross-protection test, synergism evaluation, mini-replicon assay, binding affinity analysis, docking simulation and mouse study. RESULTS: Several PB2 cap-binding inhibitors were discovered. The compound 7-(4-hydroxy-2-oxo-2H-chromen-3-yl)-6H,7H,8H-chromeno[3',4':5,6]pyrano[3,2-c]chromene-6,8-dione, designated PB2-39, was identified as a potent inhibitor of replication of multiple subtypes of influenza A virus, including H1N1, H3N2, H5N1, H7N7, H7N9 and H9N2 in vitro and H1N1, H5N1 and H7N9 in vivo. Combinational treatment with the influenza virus release inhibitor zanamivir and PB2-39 exerted a synergistic anti-influenza effect. Mechanistic experiments supported that PB2-39 suppressed viral polymerase activity. Docking and binding affinity analyses demonstrated that PB2-39 interacted with the PB2 cap-binding pocket, suggesting its role as a cap-binding competitor. CONCLUSIONS: Our study provides new insights for the strategic development of novel cap-binding inhibitors of influenza A viruses.

Glycyrrhizic Acid Reduces Heart Rate and Blood Pressure by a Dual Mechanism.

Beta adrenergic receptors are crucial for their role in rhythmic contraction of heart along with their role in the pathological conditions such as tachycardia and high risk of heart failure. Studies report that the levels of beta-1 adrenergic receptor tend to decrease by 50%, whereas, the levels of beta-2 adrenergic receptor remains constant during the risk of heart failure. Beta blockers-the antagonistic molecules for beta-adrenergic receptors, function by slowing the heart rate, which thereby allows the left ventricle to fill completely during tachycardia incidents and hence helps in blood pumping capacity of heart and reducing the risk of heart failure. In the present study, we investigate the potential of glycyrrhizic acid (GA) as a possible principal drug molecule for cardiac arrhythmias owing to its ability to induce reduction in the heart rate and blood pressure. We use in vitro and in silico approach to study GA's effect on beta adrenergic receptor along with an in vivo study to examine its effect on heart rate and blood pressure. Additionally, we explore GA's proficiency in eliciting an increase in the plasma levels of vasoactive intestinal peptide, which by dilating the blood vessel consequently, can be a crucial aid during the occurrence of a potential heart attack. Therefore, we propose GA as a potential principal drug molecule via its potential in modulating heart rate and blood pressure.

Cross-protection of influenza A virus infection by a DNA aptamer targeting the PA endonuclease domain.

Amino acid residues in the N-terminal of the PA subunit (PAN) of the influenza A virus polymerase play critical roles in endonuclease activity, protein stability, and viral RNA (vRNA) promoter binding. In addition, PAN is highly conserved among different subtypes of influenza virus, which suggests PAN to be a desired target in the development of anti-influenza agents. We selected DNA aptamers targeting the intact PA protein or the PAN domain of an H5N1 virus strain using systematic evolution of ligands by exponential enrichment (SELEX). The binding affinities of selected aptamers were measured, followed by an evaluation of in vitro endonuclease inhibitory activity. Next, the antiviral effects of enriched aptamers against influenza A virus infections were examined. A total of three aptamers targeting PA and six aptamers targeting PAN were selected. Our data demonstrated that all three PA-selected aptamers neither inhibited endonuclease activity nor exhibited antiviral efficacy, whereas four of the six PAN-selected aptamers inhibited both endonuclease activity and H5N1 virus infection. Among the four effective aptamers, one exhibited cross-protection against infections of H1N1, H5N1, H7N7, and H7N9 influenza viruses, with a 50% inhibitory concentration (IC50) of around 10 nM. Notably, this aptamer was identified at the 5th round but disappeared after the 10th round of selection, suggesting that the identification and evaluation of aptamers at early rounds of selection may be highly helpful for screening effective aptamers. Overall, our study provides novel insights for screening and developing effective aptamers for use as anti-influenza drugs.

Secretin receptor-knockout mice are resistant to high-fat diet-induced obesity and exhibit impaired intestinal lipid absorption.

Secretin, a classical gastrointestinal hormone released from S cells in response to acid and dietary lipid, regulates pleiotropic physiological functions, such as exocrine pancreatic secretion and gastric motility. Subsequent to recently proposed revisit on secretin's metabolic effects, we have confirmed lipolytic actions of secretin during starvation and discovered a hormone-sensitive lipase-mediated mechanistic pathway behind. In this study, a 12 wk high-fat diet (HFD) feeding to secretin receptor-knockout (SCTR(-/-)) mice and their wild-type (SCTR(+/+)) littermates revealed that, despite similar food intake, SCTR(-/-) mice gained significantly less weight (SCTR(+/+): 49.6±0.9 g; SCTR(-/-): 44.7±1.4 g; P<0.05) and exhibited lower body fat content. These SCTR(-/-) mice have corresponding alleviated HFD-associated hyperleptinemia and improved glucose/insulin tolerance. Further analyses indicate that SCTR(-/-) have impaired intestinal fatty acid absorption while having similar energy expenditure and locomotor activity. Reduced fat absorption in the intestine is further supported by lowered postprandial triglyceride concentrations in circulation in SCTR(-/-) mice. In jejunal cells, transcript and protein levels of a key fat absorption regulator, cluster of differentiation 36 (CD36), was reduced in knockout mice, while transcript of Cd36 and fatty-acid uptake in isolated enterocytes was stimulated by secretin. Based on our findings, a novel positive feedback pathway involving secretin and CD36 to enhance intestinal lipid absorption is being proposed.

Transmembrane peptides as unique tools to demonstrate the in vivo action of a cross-class GPCR heterocomplex.

Angiotensin (ANGII) and secretin (SCT) share overlapping, interdependent osmoregulatory functions in brain, where SCT peptide/receptor function is required for ANGII action, yet the molecular basis is unknown. Since receptors for these peptides (AT1aR, SCTR) are coexpressed in osmoregulatory centers, a possible mechanism is formation of a cross-class receptor heterocomplex. Here, we demonstrate such a complex and its functional importance to modulate signaling. Association of AT1aR with SCTR reduced ability of SCT to stimulate cyclic adenosine monophosphate (cAMP), with signaling augmented in presence of ANGII or constitutively active AT1aR. Several transmembrane (TM) peptides of these receptors were able to affect their conformation within complexes, reducing receptor BRET signals. AT1aR TM1 affected only formation and activity of the heterocomplex, without effect on homomers of either receptor, and reduced SCT-stimulated cAMP responses in cells expressing both receptors. This peptide was active in vivo by injection into mouse lateral ventricle, thereby suppressing water-drinking behavior after hyperosmotic shock, similar to SCTR knockouts. This supports the interpretation that active conformation of AT1aR is a key modulator of cAMP responses induced by SCT stimulation of SCTR. The SCTR/AT1aR complex is physiologically important, providing differential signaling to SCT in settings of hyperosmolality or food intake, modulated by differences in levels of ANGII.

Lipolytic actions of secretin in mouse adipocytes.

Secretin (Sct), a classical gut hormone, is now known to play pleiotropic functions in the body including osmoregulation, digestion, and feeding control. As Sct has long been implicated to regulate metabolism, in this report, we have investigated a potential lipolytic action of Sct. In our preliminary studies, both Sct levels in circulation and Sct receptor (SctR) transcripts in adipose tissue were upregulated during fasting, suggesting a potential physiological relevance of Sct in regulating lipolysis. Using SctR knockout and Sct knockout mice as controls, we show that Sct is able to stimulate lipolysis in vitro in isolated adipocytes dose- and time-dependently, as well as acute lipolysis in vivo. H-89, a protein kinase A (PKA) inhibitor, was found to attenuate lipolytic effects of 1 µM Sct in vitro, while a significant increase in PKA activity upon Sct injection was observed in the adipose tissue in vivo. Sct was also found to stimulate phosphorylation at 660(ser) of hormone sensitive lipase (HSL) and to bring about the translocation of HSL from cytosol to the lipid droplet. In summary, our data demonstrate for the first time the in vivo and in vitro lipolytic effects of Sct, and that this function is mediated by PKA and HSL.

Neuron-restrictive silencer factor functions to suppress Sp1-mediated transactivation of human secretin receptor gene.

In the present study, a functional neuron restrictive silencer element (NRSE) was initially identified in the 5' flanking region (-83 to -67, relative to ATG) of human secretin receptor (hSCTR) gene by promoter assays coupled with scanning mutation analyses. The interaction of neuron restrictive silencer factor (NRSF) with this motif was later indicated via gel mobility shift and ChIP assays. The silencing activity of NRSF was confirmed by over-expression and also by shRNA knock-down of endogenous NRSF. These studies showed an inverse relationship between the expression levels of NRSF and hSCTR in the cells. As hSCTR gene was previously shown to be controlled by two GC-boxes which are regulated by the ratio of Sp1 to Sp3, in the present study, the functional interactions of NRSF and Sp proteins to regulate hSCTR gene was investigated. By co-immunoprecipitation assays, we found that NRSF could be co-precipitated with Sp1 as well as Sp3 in PANC-1 cells. Interestingly, co-expressions of these factors showed that NRSF could suppress Sp1-mediated, but not Sp3-mediated, transactivation of hSCTR. Taken together, we propose here that the down-regulatory effects of NRSF on hSCTR gene expression are mediated via its suppression on Sp1-mediated transactivation.