Christoph Ullmer

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 5HT1 dβ, 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‐HT1dβ, 5‐HT2b, and 5‐HT4 receptor mRNA, whereas in smooth muscle cells 5‐HT1dβ, 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.

The membrane-bound bile acid receptor TGR5 is localized in the epithelium of human gallbladders.

TGR5 (Gpbar‐1) is a plasma membrane‐bound, G protein–coupled receptor for bile acids. TGR5 messenger RNA (mRNA) has been detected in many tissues, including rat cholangiocytes and mouse gallbladder. A role for TGR5 in gallstone formation has been suggested, because TGR5 knockout mice did not develop gallstones when fed a lithogenic diet. In this study, expression and localization of TGR5 was studied in human gallbladders. TGR5 mRNA and protein were detected in all 19 gallbladders. Although TGR5 mRNA was significantly elevated in the presence of gallstones, no such relation was found for TGR5 protein levels. In order to study the localization of TGR5 in human gallbladders, a novel antibody was generated. The receptor was localized in the apical membrane and the rab11‐positive recycling endosome of gallbladder epithelial cells. Furthermore, the TGR5 staining colocalized with the cyclic adenosine monophosphate–regulated chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) and the apical sodium‐dependent bile salt uptake transporter, suggesting a functional coupling of TGR5 to bile acid uptake and chloride secretion. Stimulation with bile acids significantly increased cyclic adenosine monophosphate concentration in human gallbladder tissue. Incubation of gallbladder epithelial cells with a TGR5 agonist led to a rise of N‐(ethoxycarbonylmethyl)‐6‐methoxyquinolinium bromide (MQAE)‐fluorescence, suggestive of a decrease in intracellular chloride concentration. The TGR5 agonist–dependent increase in MQAE‐fluorescence was absent in TGR5 knockout mice or in the presence of a CFTR inhibitor, indicating that TGR5 mediates chloride secretion via activation of CFTR. The presence of the receptor in both the plasma membrane and the recycling endosome indicate that TGR5 can be regulated by translocation. Conclusion: The data suggest a role for TGR5 in bile acid–induced fluid secretion in biliary epithelial cells. (HEPATOLOGY 2009.)

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.

Synaptic multiprotein complexes associated with 5‐HT2C receptors: a proteomic approach

Membrane‐bound receptors such as tyrosine kinases and ionotropic receptors are associated with large protein networks structured by protein–protein interactions involving multidomain proteins. Although these networks have emerged as a general mechanism of cellular signalling, much less is known about the protein complexes associated with G‐protein‐coupled receptors (GPCRs). Using a proteomic approach based on peptide affinity chromatography followed by mass spectrometry and immunoblotting, we have identified 15 proteins that interact with the C‐ terminal tail of the 5‐hydroxytryptamine 2C (5‐HT2C) receptor, a GPCR. These proteins include several synaptic multidomain proteins containing one or several PDZ domains (PSD95 and the proteins of the tripartite complex Veli3–CASK–Mint1), proteins of the actin/spectrin cytoskeleton and signalling proteins. Coimmunoprecipitation experiments showed that 5‐HT2C receptors interact with PSD95 and the Veli3–CASK–Mint1 complex in vivo. Electron microscopy also indicated a synaptic enrichment of Veli3 and 5‐HT2C receptors and their colocalization in microvilli of choroidal cells. These results indicate that the 5‐HT2C receptor is associated with protein networks that are important for its synaptic localization and its coupling to the signalling machinery.

Distinct claudins and associated PDZ proteins form different autotypic tight junctions in myelinating Schwann cells.

The apposed membranes of myelinating Schwann cells are joined by several types of junctional specializations known as autotypic or reflexive junctions. These include tight, gap, and adherens junctions, all of which are found in regions of noncompact myelin: the paranodal loops, incisures of Schmidt-Lanterman, and mesaxons. The molecular components of autotypic tight junctions have not been established. Here we report that two homologues of Discs Lost–multi PDZ domain protein (MUPP)1, and Pals-associated tight junction protein (PATJ), are differentially localized in myelinating Schwann cells and associated with different claudins. PATJ is mainly found at the paranodal loops, where it colocalized with claudin-1. MUPP1 and claudin-5 colocalized in the incisures, and the COOH-terminal region of claudin-5 interacts with MUPP1 in a PSD-95/Disc Large/zona occludens (ZO)-1 (PDZ)-dependent manner. In developing nerves, claudin-5 and MUPP1 appear together in incisures during the first postnatal week, suggesting that they coassemble during myelination. Finally, we show that the incisures also contain four other PDZ proteins that are found in epithelial tight junctions, including three membrane-associated guanylate-kinase proteins (membrane-associated guanylate-kinase inverted-2, ZO-1, and ZO-2) and the adaptor protein Par-3. The presence of these different tight junction proteins in regions of noncompact myelin may be required to maintain the intricate cytoarchitecture of myelinating Schwann cells.

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.

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 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‐HT1Dβ, 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.

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.

The membrane-bound bile acid receptor TGR5 (Gpbar-1) is localized in the primary cilium of cholangiocytes

Abstract Cholangiocyte cilia are sensory organelles that extend from the apical membrane into the bile duct lumen and detect changes in bile flow and osmolarity. Whether or not cholangiocyte cilia are responsive to bile acids is unknown. TGR5 (Gpbar-1) is a membrane-bound bile acid receptor which is expressed in biliary epithelial cells and promotes chloride secretion in gallbladder epithelial cells. As shown in the present study, TGR5 is localized in the primary cilium of mouse and human cholangiocytes. Here the receptor could play an important role in coupling biliary bile acid concentration and composition to ductular bile formation.

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‐HT1Dβ, 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‐HT1Dβ 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‐HT1Dβ 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.