Systemic bile acid sensing by G protein-coupled bile acid receptor 1 (GPBAR1) promotes PYY and GLP-1 release.

BACKGROUND AND PURPOSE: Nutrient sensing in the gut is believed to be accomplished through activation of GPCRs expressed on enteroendocrine cells. In particular, L-cells located predominantly in distal regions of the gut secrete glucagon-like peptide 1 (GLP-1) and peptide tyrosine-tyrosine (PYY) upon stimulation by nutrients and bile acids (BA). The study was designed to address the mechanism of hormone secretion in L-cells stimulated by the BA receptor G protein-coupled bile acid receptor 1 (GPBAR1). EXPERIMENTAL APPROACH: A novel, selective, orally bioavailable, and potent GPBAR1 agonist, RO5527239, was synthesized in order to investigate L-cell secretion in vitro and in vivo in mice and monkey. In analogy to BA, RO5527239 was conjugated with taurine to reduce p.o. bioavailability yet retaining its potency. Using RO5527239 and tauro-RO5527239, the acute secretion effects on L-cells were addressed via different routes of administration. KEY RESULTS: GPBAR1 signalling triggers the co-secretion of PYY and GLP-1, and leads to improved glucose tolerance. The strong correlation of plasma drug exposure and plasma PYY levels suggests activation of GPBAR1 from systemically accessible compartments. In contrast to the orally bioavailable agonist RO5527239, we show that tauro-RO5527239 triggers PYY release only when applied intravenously. Compared to mice, a slower and more sustained PYY secretion was observed in monkeys. CONCLUSION AND IMPLICATIONS: Selective GPBAR1 activation elicits a strong secretagogue effect on L-cells, which primarily requires systemic exposure. We suggest that GPBAR1 is a key player in the intestinal proximal-distal loop that mediates the early phase of nutrient-evoked L-cell secretion effects.

Functional monoclonal antibody acts as a biased agonist by inducing internalization of metabotropic glutamate receptor 7.

BACKGROUND AND PURPOSE: The mGlu(7) receptors are strategically located at the site of vesicle fusion where they modulate the release of the main excitatory and inhibitory neurotransmitters. Consequently, they are implicated in the underlying pathophysiology of CNS diseases such as epilepsy and stress-related psychiatric disorders. Here, we characterized a selective, potent and functional anti-mGlu(7) monoclonal antibody, MAB1/28, that triggers receptor internalization. EXPERIMENTAL APPROACH: MAB1/28's activity was investigated using Western blot and direct immunofluorescence on live cells, in vitro pharmacology by functional cAMP and [(35) S]-GTPγ binding assays, the kinetics of IgG-induced internalization by image analysis, and the activation of the ERK1/2 by elisa. KEY RESULTS: mGlu(7) /mGlu(6) chimeric studies located the MAB1/28 binding site at the extracellular amino-terminus of mGlu(7) . MAB1/28 potently antagonized both orthosteric and allosteric agonist-induced inhibition of cAMP accumulation. The potency of the antagonistic actions was similar to the potency in triggering receptor internalization. The internalization mechanism occurred via a pertussis toxin-insensitive pathway and did not require Gα(i) protein activation. MAB1/28 activated ERK1/2 with potency similar to that for receptor internalization. The requirement of a bivalent receptor binding mode for receptor internalizations suggests that MAB1/28 modulates mGlu(7) dimers. CONCLUSIONS AND IMPLICATIONS: We obtained evidence for an allosteric-biased agonist activity triggered by MAB1/28, which activates a novel IgG-mediated GPCR internalization pathway that is not utilized by small molecule, orthosteric or allosteric agonists. Thus, MAB1/28 provides an invaluable biological tool for probing mGlu(7) function and selective activation of its intracellular trafficking.

Discovery of potent and selective histamine H3 receptor inverse agonists based on the 3,4-dihydro-2H-pyrazino[1,2-a]indol-1-one scaffold.

A novel series of potent histamine H(3) receptor inverse agonists based on the 3,4-dihydro-2H-pyrazino[1,2-a]indol-1-one scaffold has been discovered. Several compounds display high selectivity over other histamine receptor subtypes and have favorable physicochemical properties, low potential for CYP450 enzyme inhibition and high metabolic stability in microsomal preparations. (R)-2-Cyclopropylmethyl-8-(1-isopropyl-piperidin-4-yloxy)-3-methyl-3,4-dihydro-2H-pyrazino[1,2-a]indol-1-one (8t) showed good in vivo efficacy after per os application in an acute rat dipsogenia model of water intake.

Expression of MUPP1 protein in mouse brain.

Localizing cell surface receptors to specific subcellular sites can be crucial for proper functioning. PDZ proteins apparently play central roles in such protein localizations. 5-HT(2C) receptors have previously been shown to interact with MUPP1, a multi PDZ domain protein, in heterologous systems and in rat choroid plexus. We now report the generation and characterization of two independent MUPP1 antisera, which recognise distinct areas of the mouse brain in agreement with previous in-situ hybridization studies. Our results indicate that MUPP1 immunoreactivity co-localizes with 5-HT(2A) or 5-HT(2C) receptor expression in all regions of the mouse brain, including the choroid plexus where 5-HT(2C) receptors are highly enriched.

Interaction of serotonin 5-hydroxytryptamine type 2C receptors with PDZ10 of the multi-PDZ domain protein MUPP1.

By using the yeast two-hybrid system, we previously isolated a cDNA clone encoding a novel member of the multivalent PDZ protein family called MUPP1 containing 13 PDZ domains. Here we report that the C terminus of the 5-hydroxytryptamine type 2C (5-HT(2C)) receptor selectively interacts with the 10th PDZ domain of MUPP1. Mutations in the extreme C-terminal SSV sequence of the 5-HT(2C) receptor confirmed that the SXV motif is critical for the interaction. Co-immunoprecipitations of MUPP1 and 5-HT(2C) receptors from transfected COS-7 cells and from rat choroid plexus verified this interaction in vivo. Immunocytochemistry revealed an SXV motif-dependent co-clustering of both proteins in transfected COS-7 cells as well as a colocalization in rat choroid plexus. A 5-HT(2C) receptor-dependent unmasking of a C-terminal vesicular stomatitis virus epitope of MUPP1 suggests that the interaction triggers a conformational change within the MUPP1 protein. Moreover, 5-HT(2A) and 5-HT(2B), sharing the C-terminal EX(V/I)SXV sequence with 5-HT(2C) receptors, also bind MUPP1 PDZ domains in vitro. The highest MUPP1 mRNA levels were found in all cerebral cortical layers, the hippocampus, the granular layer of the dentate gyrus, as well as the choroid plexus, where 5-HT(2C) receptors are highly enriched. We propose that MUPP1 may serve as a multivalent scaffold protein that selectively assembles and targets signaling complexes.

Parkin expression in the adult mouse brain.

Mutations in a protein designated Parkin were shown to be involved in the pathogenesis of autosomal recessive juvenile parkinsonism. Nothing is known about its regional and subcellular distribution in the mouse. In order to elucidate the Parkin mRNA and protein distribution in the adult mouse, the mouse cDNA was cloned and polyclonal antisera were generated against the N-terminal part of mouse Parkin. The antibodies were shown to be specific using Western blot analysis, immunostaining of cells transfected with mouse Parkin and pre-absorption tests. The Parkin protein expression profile was studied using immunohistochemistry and Western blot analysis and was compared with that of the mRNA yielded by in situ hybridization and RT-PCR analysis. Parkin protein was widely distributed in all subdivisions of the mouse brain. Low levels were found in the telencephalon and diencephalon, while the brainstem contained a large number of cells heavily expressing Parkin. Ultrastructural analysis and double immunohistochemistry revealed that the majority of Parkin-expressing cells were neurons, while only single glial cells exhibited immunostaining. The protein was distributed nonhomogeneously throughout the entire cytoplasm. A subpopulation of Parkin-immunopositive cells displayed speckled immunodeposits in the nucleus. Dopaminergic cells of the substantia nigra pars compacta exhibited high levels of Parkin mRNA but no Parkin protein, while the striatum contained immunopositive profiles but no mRNA signals. Our data indicate that Parkin is neither restricted to a single functional system nor associated with a particular transmitter system. The speckled nuclear distribution of Parkin immunoreactivity strongly suggests a role for Parkin in gene expression.

Cloning and characterization of MUPP1, a novel PDZ domain protein.

Using the yeast two-hybrid system we isolated a cDNA clone encoding a novel protein interacting with the C-terminal domain of the 5-HT2C receptor. The protein, named MUPP1 (multi-PDZ-domain protein), contains thirteen PDZ domains and no obvious catalytic domain; it is related to hINADL and a putative C. elegans polypeptide referred to as C52A11.4 containing six or ten PDZ domains, respectively. Domains highly similar to those of MUPP1 are arrayed in the same order in all three proteins. The MUPP1 gene is localized on human chromosome 9p24-p22. Transcripts encoding MUPP1 are abundant in the brain as well as in several peripheral organs.

Doxycycline-mediated quantitative and tissue-specific control of gene expression in transgenic mice.

The tet regulatory system in which doxycycline (dox) acts as an inducer of specifically engineered RNA polymerase II promoters was transferred into transgenic mice. Tight control and a broad range of regulation spanning up to five orders of magnitude were monitored dependent on the dox concentration in the water supply of the animals. Administration of dox rapidly induces the synthesis of the indicator enzyme luciferase whose activity rises over several orders of magnitude within the first 4 h in some organs. Induction is complete after 24 h in most organs analyzed. A comparable regulatory potential was revealed with the tet regulatory system where dox prevents transcription activation. Directing the synthesis of the tetracycline-controlled transactivator (tTA) to the liver led to highly specific regulation in hepatocytes where, in presence of dox, less than one molecule of luciferase was detected per cell. By contrast, a more than 10(5)-fold activation of the luciferase gene was observed in the absence of the antibiotic. This regulation was homogeneous throughout but stringently restricted to hepatocytes. These results demonstrate that both tetracycline-controlled transcriptional activation systems provide genetic switches that permit the quantitative control of gene activities in transgenic mice in a tissue-specific manner and, thus, suggest possibilities for the generation of a novel type of conditional mutants.

Distribution of 5-HT4 receptor mRNA in the rat brain.

We have analyzed the brain distribution of the rat 5-HT4 receptor mRNA. A receptor specific probe was used for in situ hybridization of rat brain sections. Abundant expression of the 5-HT4 receptor mRNA was observed in the olfactory system, striatum, medial habenula and the hippocampal formation, while faint or no specific signals could be detected in most other areas of the brain. Several brain areas which display strong ligand binding do not contain mRNA, suggesting an axonal localization of the 5-HT4 receptor.

Activation of meningeal 5-HT2B receptors: an early step in the generation of migraine headache?

Several pharmaceuticals are frequently dispensed to prevent or reduce the occurrence of migraine attacks. The prophylactic effect of these drugs has been suggested to be caused through blockade of serotonin (5-HT) receptors of type 5-HT2B or 5-HT2C. To elucidate which of these receptors is involved, we first used radioligand binding assays to determine the pharmacological profile of the human and rat-5-HT2B receptor. Furthermore, the potency of drugs used in migraine prophylaxis to stimulate or inhibit 5-HT2B or 5-HT2C receptor-mediated potency of drugs used in migraine prophylaxis to stimulate or inhibit 5-HT2B or 5-HT2C receptor-mediated phosphatidyl inositol hydrolysis was measured. All these drugs were found to block both human receptors. Correlation of the receptor affinities with the potencies used in migraine prophylaxis showed significant correlations, which were better for the 5-HT2B (P = 0.001) than for the 5-HT2C receptor (P = 0.005). Migraine headache is thought to be transmitted by the trigeminal nerve from the meninges and their blood vessels. Using the reverse transcription-polymerase chain reaction, the expression patterns of all cloned G-protein-coupled serotonin receptors were analysed in various human meningeal tissues. All tissues expressed 5-HT1Dbeta, 5-HT2A, 5-HT2B, 5-HT4 and 5-HT7 mRNAs. Only trace amounts of 5-HT2C receptor mRNA were found. With organ bath experiments we showed that the 5-HT2B receptor stimulated the relaxation of the pig cerebral artery via the release of nitric oxide. Our data support the hypothesis that 5-HT2B receptors located on endothelial cells of meningeal blood vessels trigger migraine headache through the formation of nitric oxide.

Functional, endogenously expressed 5-hydroxytryptamine 5-ht7 receptors in human vascular smooth muscle cells.

Human uterine artery smooth muscle cells in culture were shown to express constitutively both 5-ht7 receptor mRNA and 5-ht7-like receptors functionally linked to cyclic AMP formation. 5-Carboxamidotryptamine (5-CT) and 5-HT enhanced forskolin-stimulated cyclic AMP accumulation in these cells, with pEC50 values of 7.12 and 6.25, sumatriptan being very weakly active. Both methiothepin (0.1 microM) and clozapine (1 microM), but not the 5-HT4-receptor antagonist, SDZ 205-557 (10 microM) antagonized the effects of 5-CT. In reverse transcriptase-polymerase chain reaction analysis, the mRNA for 5-ht7, but not for 5-HT4 or 5-ht6 receptors was found to be strongly expressed in the same cells. These findings represent a further step toward the recognition of 5-ht7 receptors as real, functional receptors.

5-HT2B receptor-mediated calcium release from ryanodine-sensitive intracellular stores in human pulmonary artery endothelial cells.

1. We have characterized the 5-hydroxytryptamine (5-HT)-induced calcium signalling in endothelial cells from the human pulmonary artery. Using RT-PCR we show, that of all cloned G-protein coupled 5-HT receptors, these cells express only 5-HT1D beta, 5-HT2B and little 5-HT4 receptor mRNA. 2. In endothelial cells 5-HT inhibits the formation of adenosine 3':5'-cyclic monophosphate (cyclic AMP) via 5-HT1D beta receptors but fails to activate phosphoinositide (PI) turnover. However, the latter pathway is strongly activated by histamine. 3. Despite the lack of detectable inositol phosphate (IP) formation in human pulmonary artery endothelial cells, 5-HT (pD2 = 5.82 +/- 0.06, n = 6) or the selective 5-HT2 agonist, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) (pD2 = 5.66 +/- 0.03, n = 7) elicited transient calcium signals comparable to those evoked by histamine (pD2 = 6.44 +/- 0.01, n = 7). Since 5-HT2A and 5-HT2C receptor mRNAs are not detectable in pulmonary artery endothelial cells, activation of 5-HT2B receptors is responsible for the transient calcium release. The calcium transients are independent of the inhibition of adenylate cyclase, since DOI does not stimulate 5-HT1D beta receptors. 4. Both, the 5-HT- and histamine-stimulated calcium signals were also observed when the cells were placed in calcium-free medium. This indicates that 5-HT triggers calcium release from intracellular stores. 5. Heparin is an inhibitor of the IP3-activated calcium release channels on the endoplasmic reticulum. Intracellular infusion of heparin through patch pipettes in voltage clamp experiments failed to block 5-HT-induced calcium signals, whereas it abolished the histamine response. This supports the conclusion that the 5-HT-induced calcium release is independent of IP3 formation. 6. Unlike the histamine response, the 5-HT response was sensitive to micromolar concentrations of ryanodine and, to a lesser extent, ruthenium red. This implies that 5-HT2B receptors trigger calcium release from a ryanodine-sensitive calcium pool. 7. It has been postulated that cyclic ADP-ribose (cADPR) is a soluble second messenger which activates ryanodine receptors. However, calcium signals similar to the 5-HT response could not be elicited by intracellular infusion with cADPR. Furthermore, the subsequent application of 5-HT or DOI elicited a calcium signal that was not affected by the above pretreatment. 8. We conclude that human 5-HT2B receptors stimulate calcium release from intracellular stores through a novel pathway, which involves activation of ryanodine receptors, and is independent of PI-hydrolysis and cADPR.

Functional serotonin 5-HT1D receptors and 5-HT1D beta receptor mRNA expression in human umbilical vein endothelial cells.

Pharmacological evidence has suggested the presence of 5-hydroxytryptamine (5-HT, serotonin), 5-HT(1D) receptors on endothelial cells but these receptors have never been identified unambiguously on this type of cells. We now report that human umbilical vein endothelial cells (HUVEC) express 5-HT(1D) receptors coupled to inhibition of cyclic AMP formation. 5-HT and 5-HT(1D) receptor agonists 5-carboxamidotryptamine (5-CT) and sumatriptan were approximately equipotent at inhibiting forskolin-stimulated cyclic AMP accumulation in HUVEC (mean pEC50 7.6-8.2, maximal effect 30% inhibition). The 5-HT(1A) receptor antagonist, 8-OH-DPAT was clearly less potent (pEC50 6.2) and less efficacious. The selective 5-HT(1D) receptor agonist, GR127935 (1 nM) markedly inhibited the effect of 5-HT (apparent pK(B) 10.8). Reverse transcription-polymerase chain reaction analysis showed the mRNA for 5-HT(1D beta) receptors to be expressed in HUVEC. These results demonstrate the presence of functional 5-HT(1D) receptors and the expression of 5-HT(1D beta) receptor mRNA in HUVEC. They support the involvement of 5-HT(1D beta) receptors in endothelial-mediated responses to 5-HT.

Expression of serotonin receptor mRNAs in blood vessels.

Using RT-PCR we distinguished mRNAs for all known G-protein coupled serotonin receptors expressed in various rat and porcine blood vessels. Nearly all vessels expressed 5HT1D beta, 5-HT2A, 5-HT2B, 5-HT4, and 5-HT7 receptor mRNA to different extents. New splice variants of the porcine 5-HT4 receptor were observed. Similar PCR assays were performed with endothelial and smooth muscle cells from human pulmonary artery, aorta, and with endothelial cells from human coronary artery and umbilical vein. All endothelial cells expressed 5-HT1D beta, 5-HT2B, and 5-HT4 receptor mRNA, whereas in smooth muscle cells 5-HT1D beta, 5-HT2A, 5-HT7, and in some experiments 5-HT2B receptor mRNA were found. A model for the regulation of vascular tone by different 5-HT receptors is proposed.

Differential transactivation potential of Oct1 and Oct2 is determined by additional B cell-specific activities.

Cell type-specific transcriptional regulation is generally believed to be mediated by sequence-specific transcription factors that are specifically present in the corresponding cells. The interaction of the lymphoid-specific Oct2 transcription factor has been thought to be responsible for the B cell-specific activity of octamer-containing promoter and enhancer elements. Here we show that physiological concentrations of Oct2 do not suffice to generate octamer-dependent promoter activity in non-B cell lines. Furthermore, we have tested the activity of octamer-dependent promoter and enhancer elements in B cell lines that lack the endogenous Oct2 protein. Our results demonstrate that in these Oct2-deficient B cells the ubiquitous endogenous Oct1 protein is able to stimulate octamer-containing promoters to a level comparable with that of normal Oct2-positive B cells. However, reporter constructs bearing the octamer motif in a distal enhancer position are not stimulated by the Oct1 protein, but do require the presence of Oct2. The B cell-specific octamer-dependent promoter activity mediated by Oct1 correlates with the presence of a novel B cell-specific octamer-binding complex containing the Oct1 protein. From these results we conclude that B cells contain two different activities: one that interacts with both Oct1 and Oct2 and mediates promoter proximal activity of the octamer motif and a second that specifically interacts with Oct2 to confer function from a remote enhancer position.

Cloning and functional characterization of the human 5-HT2B serotonin receptor.

Recently, we have reported the cloning of the rat 5-HT2B receptor cDNA. This receptor is particularly interesting since it may be involved in diseases such as migraine. Here, we describe the isolation of a human 5-HT2B receptor clone from a cDNA library derived from SH-SY5Y cells. Although the receptor sequence was only 80% homologous to the rat sequence, the exon-intron distribution was conserved between the two species. In the human body, the receptor mRNA was detected in most peripheral organs. Only low expression levels were found in the brain. After expression in HEK 293 cells, activation of the receptor stimulated the production of phosphatidylinositol. The pharmacology of this functional response correlated well with that of the rodent receptor.