ELABELA (ELA) was recently discovered as a novel endogenous ligand of the apelin receptor (APJ), a G protein-coupled receptor. ELA signaling was demonstrated to be crucial for normal heart and vasculature development during embryogenesis. We delineate here ELA's structure-activity relationships and report the identification of analogue 3 (ELA(19-32)), a fragment of ELA that binds to APJ, activates the Gαi1 and β-arrestin-2 signaling pathways, and induces receptor internalization similarly to its parent endogenous peptide. An alanine scan performed on 3 revealed that the C-terminal residues are critical for binding to APJ and signaling. Finally, using isolated-perfused hearts and in vivo hemodynamic and echocardiographic measurements, we demonstrate that ELA and 3 both reduce arterial pressure and exert positive inotropic effects on the heart. Altogether, these results present ELA and 3 as potential therapeutic options in managing cardiovascular diseases.

Activation of the apelinergic system by exogenous ELA or Apelin-13 infusion improves cardiovascular function and survival after cecal ligation puncture-induced sepsis. However, ELA proved better than Apelin-13 by improving fluid homeostasis, cardiovascular hemodynamics recovery, and limiting kidney dysfunction in a vasopressinergic-dependent manner.

Epoxyeicosatrienoic acids (EETs) are produced from arachidonic acid via the cytochrome P-450 epoxygenase pathway. EETs are able to modulate smooth muscle tone by increasing K(+) conductance, hence generating hyperpolarization of the tissues. However, the molecular mechanisms by which EETs induce smooth muscle relaxation are not fully understood. In the present study, the effects of EETs on airway smooth muscle (ASM) were investigated using three electrophysiological techniques. 8,9-EET and 14,15-EET induced concentration-dependent relaxations of the ASM precontracted with a muscarinc agonist (carbamylcholine chloride), and these relaxations were partly inhibited by 10 nM iberiotoxin (IbTX), a specific large-conductance Ca(2+)-activated K(+) (BK(Ca)) channel blocker. Moreover, 3 microM 8,9- or 14,15-EET induced hyperpolarizations of -12 +/- 3.5 and -16 +/- 3 mV, with EC(50) values of 0.13 and 0.14 microM, respectively, which were either reversed or blocked on addition of 10 nM IbTX. These results indicate that BK(Ca) channels are involved in hyperpolarization and participate in the relaxation of ASM. In addition, complementary experiments demonstrated that 8,9- and 14,15-EET activate reconstituted BK(Ca) channels at low free Ca(2+) concentrations without affecting their unitary conductance. These increases in channel activity were IbTX sensitive and correlated well with the IbTX-sensitive hyperpolarization and relaxation of ASM. Together these results support the view that, in ASM, the EETs act through an epithelium-derived hyperpolarizing factorlike effect.

The pregnane X receptor (PXR) is a master regulator of xenobiotic clearance and is implicated in deleterious drug interactions (e.g., acetaminophen hepatotoxicity) and cancer drug resistance. However, small-molecule targeting of this receptor has been difficult; to date, directed synthesis of a relatively specific PXR inhibitor has remained elusive. Here we report the development and characterization of a first-in-class novel -12)] that antagonizes the activated state of PXR with limited effects on other related nuclear receptors (i.e., liver X receptor, farnesoid X receptor, estrogen receptor ␣, peroxisome proliferator-activated receptor ␥, and mouse constitutive androstane receptor). We investigated the toxicity and PXR antagonist effect of FLB-12 in vivo. Compared with ketoconazole, a prototypical PXR antagonist, FLB-12 is significantly less toxic to hepatocytes. FLB-12 significantly inhibits the PXR-activated loss of righting reflex to 2,2,2-tribromoethanol (Avertin) in vivo, abrogates PXRmediated resistance to 7-ethyl-10-hydroxycamptothecin (SN-38) in colon cancer cells in vitro, and attenuates PXR-mediated acetaminophen hepatotoxicity in vivo. Thus, relatively selective targeting of PXR by antagonists is feasible and warrants further investigation. This class of agents is suitable for development as chemical probes of PXR function as well as potential PXRdirected therapeutics.

Clinically, nitric oxide (NO*) is widely used as a pulmonary vaso- and bronchodilator agent. However, the precise molecular mechanisms by which NO. induces smooth muscle relaxation are not well established. It has been suggested that NO. relaxes airway smooth muscle (ASM) via a 3',5'-cyclic guanosine monophosphate (cGMP)-dependent pathway, and our previous work has shown that Ca2+-activated K+ (KCa) channels are susceptible to cGMP-dependent protein kinase (PKG)-dependent phosphorylation (A. Alioua, J. P. Huggins, and E. Rousseau. Am. J. Physiol. 1995;268:L1057-L1063). To assess whether KCa channels are also directly activated by NO. or one of its derivatives such as peroxynitrite, the activity of these channels was measured upon fusion of sarcolemmal vesicles derived from bovine tracheal smooth muscle cells into planar lipid bilayers (PLB). It was found that in the absence of adenosine triphosphate (ATP), cGMP, and cGMP-dependent protein kinase, NO* donors such as 1-propanamine-3-(2-hydroxy-2-nitroso-1-propylhydrazine) (PAPA NONOate) or 3-morpholinosydnonimine hydrochloride (SIN-1) in the presence of superoxide dismutase (SOD), added on either side of the bilayer, caused a concentration- dependent increase in the open probability (Po) of KCa channels without altering their unitary conductance. Release of NO*, which was measured by chemiluminescence analysis in parallel experiments, affected the gating behavior of KCa channels in the presence of SOD and ethyleneglycol-bis-(beta-aminoethyl ether)- N,N'-tetraacetic acid (EGTA) by reducing the mean closed times and increasing the number and duration of short open events. PAPA NONOate, a true NO. donor, had similar effects in the presence of ethylenediaminetetraacetic acid (EDTA), a heavy-metal chelator, and K-urate, a peroxynitrite scavenger. Addition of either 5 mM dithiothreitol (DTT) or 5 mM reduced glutathione (GSH), as well as 5 mM N-ethylmaleimide (NEM)-an alkylating agent-to the trans (intracellular) side of an experimental chamber slightly increased channel Po but prevented further channel activation by NO* donors. However, neither DTT nor GSH was able to reverse the effect of NO*. In contrast to SIN-1, DTT had no effect when added to the cis (extracellular) side of the chamber. This suggests that the effect of NO* is most likely due to a chemical modification (nitrothiosylation) of intracellular sulfhydryl group(s). Neither PAPA NONOate (NO*), nor SIN-1 had any effect on sarcolemmal Cl- channels reconstituted from the same membrane preparations. Pharmacomechanical measurements made on epithelium-denuded rat bronchus showed that 100 nM charybdotoxin decreased the sensitivity of bronchial smooth muscle to SIN-1-induced relaxations. Altogether, our data suggest that NO-induced bronchorelaxation occurs partly via a direct activation of KCa channels, possibly through a covalent interaction with the cytoplasmic side of their alpha subunit.