Hsiao-Ling Wu

Neuromedin U Is a Potent Agonist at the Orphan G Protein-coupled Receptor FM3

Neuromedins are a family of peptides best known for their contractile activity on smooth muscle preparations. The biological mechanism of action of neuromedin U remains unknown, despite the fact that the peptide was first isolated in 1985. Here we show that neuromedin U potently activates the orphan G protein-coupled receptor FM3, with subnanomolar potency, when FM3 is transiently expressed in human HEK-293 cells. Neuromedins B, C, K, and N are all inactive at this receptor. Quantitative reverse transcriptase-polymerase chain reaction analysis of neuromedin U expression in a range of human tissues showed that the peptide is highly expressed in the intestine, pituitary, and bone marrow, with lower levels of expression seen in stomach, adipose tissue, lymphocytes, spleen, and the cortex. Similar analysis of FM3 expression showed that the receptor is widely expressed in human tissue with highest levels seen in adipose tissue, intestine, spleen, and lymphocytes, suggesting that neuromedin U may have a wide range of presently undetermined physiological effects. The discovery that neuromedin U is an endogenous agonist for FM3 will significantly aid the study of the full physiological role of this peptide.

Human AT1 receptor is a single copy gene: Characterization in a stable cell line

To address conflicting reports concerning the number of angiotensin II (AII) receptor type 1 (AT1) coding loci in vertebrates, Southern blot analysis was used to determine the genomic representation of AT1 receptor genes in animals comprising a divergent evolutionary spectrum. The data demonstrate that the AT1 receptor gene is present as a single genomic copy in a broad spectrum of animals including human, monkey, dog, cow, rabbit, and chicken. In contrast, members of the rodent taxonomic order contain two genes in their genomes. These two genes may have arisen in rodents as a consequence of a gene duplication event that occurred during evolution following the branching of rodents from the mammalian phylogenetic tree. In order to investigate the properties of the human AT1 receptor in a pure cell system, the recombinant human AT1 receptor was stably expressed in mouse L cells. An isolated cell line, designated LhAT1-D6, was found to express abundant levels of recombinant receptor [430±15 fmol/mg] exhibiting high affinity [KD=0.15±0.02 nM] for [125I][SAR1, IIe8] angiotensin II (SIA). The pharmacological profile of ligands competing for [125I] SIA binding to the expressed recèptor was in accordance with that of the natural receptor. Radioligand binding of the expressed receptor was decreased in the presence of the non-hydrolyzable analog of GTP, guanosine 5′-(γ-thio) triphosphate [GTPγS]. Angiotensin II evoked a rapid efflux of45Ca2+ from LhAT1-D6 cells that was blocked by AT1 receptor specific antagonists. In addition, AII inhibited forskolin-stimulated cAMP accumulation in these cells which was blocked by the AT-1 antagonist. Thus, the LhAT1-D6 cell line provides a powerful tool to explore the human AT1 receptor regulation.

Molecular Characterization of a Novel Human Endothelin Receptor Splice Variant

Endothelin receptors are widely distributed throughout a number of tissues. A novel ETB receptor splice variant (ETB-SVR) was identified from a human placental cDNA library. Sequence analysis indicated that the ETB-SVR is 436 amino acids long and shares 91% identity to the human ETB-R. Northern blot analysis indicated an mRNA species of 2.7 kilobases, which is expressed in the lung, placenta, kidney, and skeletal muscle. Ligand binding studies of the cloned ETB-SVR and ETB-R receptors expressed in COS cells showed that ET peptides exhibited similar potency in displacing 125I-ET-1 binding. Functional studies showed that ET-1, ET-3, and sarafotoxin 6c displayed similar potencies for inositol phosphates accumulation in ETB-R-transfected COS cells, whereas no increase in inositol phosphate accumulation was observed in ETB-SVR-transfected cells. In addition, exposure of ETB-R-transfected cells to ET-1 caused an increase in the intracellular acidification rate whereas ETB-SVR-transfected cells did not respond to ET-1. These data suggest that the ETB-SVR and ETB-R are functionally distinct and the difference in the amino acid sequences between the two receptors may determine functional coupling. Availability of cDNA clones for endothelin receptors can facilitate our understanding of the role of ET in the pathophysiology of various diseases.

Characterization of the leukotriene D4 receptor in guinea-pig lung

The effects of monovalent, divalent cations, buffere species and pH dependency on [3H]leukotriene D4 binding to the receptor have been characterized in vitro by using a radioligand binding assay. It was found that Ca2+, Mg2+, Co2+ and Mn2+ enhanced the specific binding. High concentrations of NaCl (150-300 mM) inhibited the specific binding to the receptor. The specific binding was also found to be higher in Pipes buffer (pH 6.5) than in Tris, Hepes and phosphate buffer at pH 7.0-8.0. Conversion of [3H]leukotriene D4 was minimized by inclusion of 1 mM cysteine, glycine in the incubation buffer and maintaining the temperature at 22 degrees C. Under the conditions employed, the dissociation constant (KD) and the receptor density (Bmax) were calculated to be 1.8 +/- 0.9 nM and 2100 +/- 375 fmol/mg protein respectively. The leukotriene antagonist FPL 55712, agonist 5R, 6S-LTD4 and LTE4 competed with the [3H]LTD4 binding to the receptor. Prostaglandins, alpha-, beta-adrenergic and dopaminergic receptor agonists and antagonists did not compete significantly.

Leukotriene E4 binds specifically to LTD4 receptors in guinea pig lung membranes

High affinity, stereoselective binding sites for [3H]leukotriene E4 ([3H]LTE4) have been identified and characterized in guinea pig lung membranes. [3H]LTE4 bound to these membranes with a pharmacological specificity identical to that previously observed for the [3H]LTD4 receptor in guinea pig lung. [3H]LTE4 specific binding was selectively inhibited by Na+, enhanced by Ca2+, Mg2+ and Mn2+ and modulated by guanine nucleotides. Scatchard analysis of saturation binding data showed a single class of high affinity and saturable binding sites, with a dissociation constant (Kd) of 0.4 +/- 0.2 nM and a density (Bmax) of 430 +/- 50 fmol/mg membrane protein, similar to values observed for the LTD4 receptor in guinea pig lung. The rank order potency of agonist binding to the [3H]LTE4 binding sites was LTD4 greater than LTE4 much greater than LTC4. These results indicate that [3H]LTE4 binds to [3H]LTD4 receptors and suggests that induction of smooth muscle contraction by LTD4 and LTE4 may be mediated by identical mechanisms and receptors in the guinea pig lung.

Identification of leukotriene D4 specific binding sites in the membrane preparation isolated from guinea pig lung

A radioligand binding assay has been established to study leukotriene specific binding sites in the guinea pig and rabbit tissues. Using high specific activity [3H]-leukotriene D4 [( 3H]-LTD4), in the presence or absence of unlabeled LTD4, the diastereoisomer of LTD4 (5R,6S-LTD4), leukotriene E4 (LTE4) and the end-organ antagonist, FPL 55712, we have identified specific binding sites for [3H]-LTD4 in the crude membrane fraction isolated from guinea pig lung. The time required for [3H]-LTD4 binding to reach equilibrium was approximately 20 to 25 min at 37 degrees C in the presence of 10 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl. The binding of [3H]-LTD4 to the specific sites was saturable, reversible and stereospecific. The maximal number of binding sites (Bmax), derived from Scatchard analysis, was approximately 320 +/- 200 fmol per mg of crude membrane protein. The dissociation constants, derived from kinetic and saturation analyses, were 9.7 nM and 5 +/- 4 nM, respectively. The specific binding sites could not be detected in the crude membrane fraction prepared from guinea pig ileum, brain and liver, or rabbit lung, trachea, ileum and uterus. In radioligand competition experiments, LTD4, FPL 55712 and 5R,6S-LTD4 competed with [3H]-LTD4. The metabolic inhibitors of arachidonic acid and SKF 88046, an antagonist of the indirectly-mediated actions of LTD4, did not significantly compete with [3H]-LTD4 at the specific binding sites. These correlations indicated that these specific binding sites may be the putative leukotriene receptors in the guinea-pig lung.

Selective inhibition of rat mesangial cell proliferation by a synthetic peptide derived from the sequence of the C2 region of PKCβ

RACK (receptor for activated C-kinase) is a protein that binds and translocates protein kinase C (PKC) to the appropriate cellular organelles. The binding of RACK has been mapped to C2 region of PKC. A number of peptides from the C2 region of PKCbeta have been shown to inhibit the translocation and activation of PKCbeta. This investigation was undertaken to study the role of PKCbeta in rat mesangial cell proliferation mediated by a number of mitogens. Exposure of rat mesangial cells to thrombin, endothelin, epidermal growth factor, and phorbol 12,13-dibutyrate resulted in increased [3H]thymidine incorporation. Pretreatment of mesangial cells with Ro 32-0432 (selective PKC inhibitor) inhibited the proliferation mediated by all the above mitogens, suggesting that these mitogens mediated proliferation through PKC. Experiments were performed to further evaluate the involvement of PKCbeta in this process by using the peptide derived from the C-2 region of PKCbeta as a tool. The data suggest that although the peptide (P) alone had no effect on basal- or mitogen-mediated proliferation, the peptide in the presence of a carrier peptide (PC) inhibited proliferation mediated by endothelin. In the same experiment, proliferation mediated by epidermal growth factor, thrombin and phorbol dibutyrate was unaffected, suggesting that in rat mesangial cells, endothelin mediated proliferation through the activation of PKCbeta.

Stimulation of Hyaluronan Synthetase by Platelet-Derived Growth Factor bb in Human Prostate Smooth Muscle Cells

Hyaluronic acid (HA) is an important component of the extracellular matrix of prostate cells. Platelet-derived growth factor bb (PDGFbb) has a mitogenic effect on prostate stromal cells. The effect of PDGFbb on HA production by smooth muscle cells from normal and benign prostatic hyperplasia (BPH) tissue was studied to elucidate the role of these two factors in BPH. The basal level of HA released into the cell media was greater in BPH cells. PDGFbb increases HA production in normal and BPH smooth muscle cells. This is partly due to an increase in HA synthetase II mRNA expression. In prostate disease, PDGFbb may have a role that involves HA production.

Transforming Growth Factor-β-Mediated Proliferation of Renal Tubular Epithelial Cells Involves Pertussis Toxin-Sensitive G Protein- and Protein Kinase C-Dependent Pathways

Exposure of LLCPK-1 cells to transforming growth factor (TGF)-β₁ resulted in an increase in [³H]thymidine incorporation. This increase depended on the concentration of TGF-β₁ used with an EC₅₀ value of 0.3–1 pmol/l. Concentrations >10 pmol/l were inhibitory. Similar effects occurred with TGF-β₃. Addition of both TGF-β₁ and TGF-β₃ together had no additive effect. Pretreatment of cells with 10 µmol/l genestein exerted no effect on TGF-β-mediated response, whereas pretreatment with 10 µmol/l Ro-32-0432 inhibited TGF-β-mediated [³H]thymidine incorporation. In addition, pretreatment of cells with pertussis toxin attenuated TGF-β-mediated proliferation of LLCPK-1 cells. These data indicate that in LLCPK-1 cells, TGF-β₁ and TGF-β₃ mediate their responses through the same receptor. This pathway involves protein kinase C and pertussis toxin-sensitive G protein.

Inhibition of endothelin-mediated topoisomerase I activation by pertussis toxin

Cultured rat mesangial cells contain high affinity endothelin (ET) receptors at high densities. Exposure of these cells to ET resulted in a transient activation of topoisomerase I extractable activity, which reached its maximum value at approximately 2 min and returned to basal value after approximately 10 min of treatment. The activation of this enzyme was dependent upon the concentration of ET added. Incubation of the cells with pertussis toxin inhibited ET‐induced increases in topoisomerase I activity in a concentration‐dependent manner, suggesting the involvement of pertussis toxin‐sensitive GTP‐binding protein in ET‐mediated action. Endothelin had no detectable effect upon extractable topoisomerase II activity.