Agonist-specific requirement for a glutamate in transmembrane helix 1 of the oxytocin receptor.

Defining key differences between agonist and antagonist binding to hormone receptors is important and will aid rational drug design. Glu(1.35) in transmembrane helix 1 (TM1) of the human oxytocin receptor (OTR) is absolutely conserved in all OTRs cloned to date. We establish that Glu(1.35) is critical for high affinity binding of agonists (full and partial) but is not required for antagonist binding (peptide or non-peptide). Consequently, the mutant receptor [E1.35A]OTR exhibited markedly decreased OT affinity (>1200-fold) and disrupted second messenger generation. Substitutions of Glu(1.35) by Asp, Gln or Arg were incapable of supporting wild-type OTR agonist binding or signaling. Molecular modeling revealed that Glu(1.35) projects into the receptor's central binding crevice and provides agonist-specific contacts not utilized by antagonists. This study explains why Glu is absolutely conserved at residue-1.35 in all receptors binding OT and related peptides, and provides molecular insight into key differences between agonist-receptor and antagonist-receptor binding modes.

Pregnane X receptor activators inhibit human hepatic stellate cell transdifferentiation in vitro.

BACKGROUND & AIMS: The activated pregnane X receptor is antifibrogenic in rodent chronic liver injury in vivo models. The aim of this study was to determine the effects of human pregnane X receptor activators on human hepatic stellate cell transdifferentiation to a profibrogenic phenotype in vitro. METHODS: Hepatic stellate cells were isolated from resected human liver and cultured under conditions in which they trans-differentiate into profibrogenic myofibroblasts. RESULTS: The pregnane X receptor was expressed in primary cultures at the level of messenger RNA and protein and was activated by the ligand rifampicin as judged by increases in binding of proteins to the pregnane X receptor ER6 DNA response element and by increases in ER6-dependent reporter gene expression. Short-term treatment of hepatic stellate cells with rifampicin inhibited the expression of selected fibrosis-related genes (transforming growth factor beta1, alpha-smooth muscle actin), proliferation-related genes, and WNT signaling-associated genes. There was also an increase in interleukin-6 secretion and an inhibition in DNA synthesis. Long-term treatment with rifampicin over several weeks reduced the proliferation and transdifferentiation of hepatic stellate cells. Small interfering RNA knockdown of the pregnane X receptor in a hepatic stellate cell line reduced the binding of proteins to the ER6 DNA response element and abrogated pregnane X receptor activator-dependent changes in transforming growth factor beta1 expression, interleukin-6 secretion, and proliferation. CONCLUSIONS: The pregnane X receptor is transcriptionally functional in human hepatic stellate cells and activators inhibit transdifferentiation and proliferation. The pregnane X receptor may therefore be an effective target for antifibrotic therapy.

Mechanism of action of the antifibrogenic compound gliotoxin in rat liver cells.

Gliotoxin has been shown to promote a reversal of liver fibrosis in an animal model of the disease although its mechanism of action in the liver is poorly defined. The effects of gliotoxin on activated hepatic stellate cells (HSCs) and hepatocytes have therefore been examined. Addition of gliotoxin (1.5 microM) to culture-activated HSCs resulted in its rapid accumulation, resulting in increased levels of glutathione and apoptosis without any evidence of oxidative stress. In contrast, although hepatocytes also rapidly sequestered gliotoxin, cell death only occurred at high (50-microM) concentrations of gliotoxin and by necrosis. At high concentrations, gliotoxin was metabolized by hepatocytes to a reduced (dithiol) metabolite and glutathione was rapidly oxidized. Fluorescent dye loading experiments showed that gliotoxin caused oxidative stress in hepatocytes. Antioxidants--but not thiol redox active compounds--inhibited both oxidative stress and necrosis in hepatocytes. In contrast, HSC apoptosis was not affected by antioxidants but was potently abrogated by thiol redox active compounds. The adenine nucleotide transporter (ANT) is implicated in mitochondrial-dependent apoptosis. HSCs expressed predominantly nonliver ANT isoform 1, and gliotoxin treatment resulted in a thiol redox-dependent alteration in ANT mobility in HSC extracts, but not hepatocyte extracts. In conclusion, these data suggest that gliotoxin stimulates the apoptosis of HSCs through a specific thiol redox-dependent interaction with the ANT. Further understanding of this mechanism of cell death will aid in finding therapeutics that specifically stimulate HSC apoptosis in the liver, a promising approach to antifibrotic therapy.

Non-peptide oxytocin agonists.

A library of compounds targeted to the vasopressin/oxytocin family of receptors was screened for activity at a cloned human oxytocin receptor using a reporter gene assay. Potency and selectivity were optimised to afford compound 39, EC50 = 33 nM. This series of compounds represents the first disclosed, non-peptide, low molecular weight agonists of the hormone oxytocin (OT).