Search

Beta-arrestins (βarrs) critically mediate desensitization, endocytosis and signalling of G protein-coupled receptors (GPCRs), and they scaffold a large number of interaction partners. However, allosteric modulation of their scaffolding abilities and direct targeting of their interaction interfaces to modulate GPCR functions selectively have not been fully explored yet. Here we identified a series of synthetic antibody fragments (Fabs) against different conformations of βarrs from phage display libraries. Several of these Fabs allosterically and selectively modulated the interaction of βarrs with clathrin and ERK MAP kinase. Interestingly, one of these Fabs selectively disrupted βarr–clathrin interaction, and when expressed as an intrabody, it robustly inhibited agonist-induced endocytosis of a broad set of GPCRs without affecting ERK MAP kinase activation. Our data therefore demonstrate the feasibility of selectively targeting βarr interactions using intrabodies and provide a novel framework for fine-tuning GPCR functions with potential therapeutic implications.

For a prototypical GPCR, the human vasopressin receptor type 2 (V2R), βarr1 can form a stable complex associated via only the phosphorylated carboxyl terminus of the receptor. Such a partially engaged complex is functionally competent with respect to supporting receptor internalization and ERK MAPK activation.

Postnatal and adult neurogenesis are region- and modality-specific, but the significance of developmentally distinct neuronal populations remains unclear. We demonstrate that chemogenetic inactivation of a subset of forebrain and olfactory neurons generated at birth disrupts responses to an aversive odor. In contrast, novel appetitive odor learning is sensitive to inactivation of adult-born neurons, revealing that developmentally defined sets of neurons may differentially participate in hedonic aspects of sensory learning.

An aneurysm is a medical condition where a section of the artery bulges out under high pressure. Patients suffering from aneurysm rupture have a mortality rate of around 20% and a morbidity rate of up to 40%. The present imaging methods, such as MRI and CT scans, only offer geometrical information on the aneurysm and cannot predict the risk of rupture associated with its progression. To address this gap, a novel computational modeling framework was developed to describe aneurysm growth and analyze the rupture risk under varying pressure loading conditions. The aneurysms were modeled at the vulnerable posterior cerebral artery (PCA) and posterior communicating artery (PCoA), extracted using image segmentation. Five different aneurysm diameters and two wall thicknesses were considered to simulate different phases of aneurysm progression. The realistic pressure loadings on the posterior cerebral arteries were described using three pressures (diastolic, systolic, and hypertensive), and the stress distributions across all models were evaluated to estimate the rupture risk. For PCA, the value of max. von-Mises stress varied between 5.334 MPa and 13.324 MPa for different models with wall thickness of 0.05 mm and from 2.579 MPa to 7.582 MPa for 0.1 mm wall thickness models. For PCoA, the value of max. von-Mises stress ranged from 2.073 MPa to 11.383 MPa for artery-aneurysm models with 0.075 mm thickness and from 2.817 MPa to 10.779 MPa for artery-aneurysm models with 0.15 mm thickness. It was found that the stress values on the aneurysm walls significantly varies with change in blood pressure and aneurysm diameter. An aneurysm with a large diameter and thin wall was also observed to pose a significant risk of rupture, particularly at high blood pressures. These results are expected to provide valuable information to the medical practitioners and help in the prediction of rupture risks using image analysis of aneurysm size and in making timely treatment decisions.

Intranasal administration can potentially deliver drugs to the brain because of the proximity of the delivery site to the olfactory lobe. We prepared triturates of micronized or crystalline zolmitriptan with a GRAS substance, nicotinamide, to form a eutectic. We characterized the formulation using differential scanning calorimetry, powder X-ray diffraction, and FTIR spectroscopy to confirm its eutectic nature and generated a phase diagram. The eutectic formulation was aerosolized using an in-house insufflator into the nares of rats. Groups of rats received zolmitriptan intravenously or intranasally, or intranasal eutectic formulation. Zolmitriptan was estimated in the olfactory lobe, cerebral cortex, cerebellum, and blood plasma at different time-points by LC–MS. Pharmacokinetics in these tissues indicated the superiority of the intranasal eutectic formulation for brain targeting when compared with results of IV solution and intranasal pure zolmitriptan powder. Enhancement of nose-to-brain transport is likely to have resulted from more rapid dissolution of the eutectic as compared to pure drug.

Bone marrow stromal cells (BMSCs) are a source of autologous stem cells that have the potential for undergoing differentiation into multiple cell types including neurons. Although the neuronal differentiation of mesenchymal stem cells has been studied for a long time, the molecular players involved are still not defined. Here we report that the genetic deletion of two members of the bone morphogenetic protein (Bmp) family, Bmp2and Bmp4in mouse BMSCs causes their differentiation into cells with neuron-like morphology. Surprisingly these cells expressed certain markers characteristic of both neuronal and glial cells. Based on this observation, we inhibited BMP signaling in mouse BMSCs through a brief exposure to Noggin protein which also led to their differentiation into cells expressing both neuronal and glial markers. Such cells seem to have the potential for further differentiation into subtypes of neuronal and glial cells and thus could be utilized for cell-based therapeutic applications.

This study identified koenidine (4) as a metabolically stable antidiabetic compound, when evaluated in a rodent type 2 model (leptin receptor-deficient db/dbmice), and showed a considerable reduction in the postprandial blood glucose profile with an improvement in insulin sensitivity. Biological studies were directed from the preliminary in vitro evaluation of the effects of isolated carbazole alkaloids (1–6) on glucose uptake and GLUT4 translocation in L6-GLUT4mycmyotubes, followed by an investigation of their activity (2–5) in streptozotocin-induced diabetic rats. The effect of koenidine (4) on GLUT4 translocation was mediated by the AKT-dependent signaling pathway in L6-GLUT4mycmyotubes. Moreover, in vivo pharmacokinetic studies of compounds 2and 4clearly showed that compound 4was 2.7 times more bioavailable than compound 2, resulting in a superior in vivo efficacy. Therefore, these studies suggested that koenidine (4) may serve as a promising lead natural scaffold for managing insulin resistance and diabetes.

Intestinal ischemia has a wide variety of causes, including, but not limited to, atherosclerosis, thrombosis, hypotension, and chronic inflammation. In severe cases, ischemic injury can result in death. μ-Opioid receptor (MOR) signaling has previously been shown to protect against chemically induced colitis, but the cellular origin of this effect remains unknown. Herein, we evaluated the role of intestinal epithelial cell (IEC)–derived MOR signaling in host responses to ischemia/reperfusion-induced injury. Ileal ischemia was accomplished through obstruction of the distal branches of the superior mesenteric artery (60 minutes) and reperfusion for 90 minutes (ischemia-reperfusion). Floxed-MOR mice were crossed to Villin-cre transgenic mice to selectively delete the MORgene in IECs (MORIEC−/−). Radio-ligand binding assays demonstrated selective loss of MOR signaling in IECs of MORIEC−/−mice. The s.c. administration of the MOR agonist, [D-Arg2, Lys4] dermorphin (1–4) amide (DALDA), 10 minutes before surgery protected against both ischemic and reperfusion phases of intestinal injury, an effect abolished in MORIEC−/−mice. This cytoprotective effect was associated with enterocyte-mediated phosphoinositide 3-kinase (PI3K)/glycogen synthase kinase 3β signaling and decreased apoptosis, as determined by IHC and caspase-3 processing. PI3K blockade with Ly294002 resulted in loss of MOR-mediated cytoprotective function. Together, these data show that IEC-derived μ-opioid signaling uses the PI3K pathway to protect cells against the damaging effect of ischemia-reperfusion. Targeting MOR signaling may represent a novel mean to alleviate intestinal injury and promote the wound-healing response.

Dysregulation of the 5-HT(2A) receptor is implicated in both the etiology and treatment of schizophrenia. Although the essential role of 5-HT(2A) receptors in atypical antipsychotic drug actions is widely accepted, the contribution of 5-HT(2A) down-regulation to their efficacy is not known. We hypothesized that down-regulation of cortical 5-HT(2A) receptors contributes to the therapeutic action of atypical antipsychotic drugs. To test this hypothesis, we assessed the effect of chronically administered antipsychotics (clozapine, olanzapine, and haloperidol) and several 5-HT(2A) antagonists [ketanserin, altanserin, α-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenylethyl)]-4-piperidinemethanol (M100907), α-phenyl-1-(2-phenylethyl)-4-piperidinemethano (M11939), 4-[(2Z)-3-{[2-(dimethylamino)ethoxy]amino}-3-(2-fluorophenyl)prop-2-en-1-ylidene]cyclohexa-2,5-dien-1-one (SR46349B), and pimavanserin], on the phencyclidine (PCP)-induced hyperlocomotor response and cortical 5-HT(2A) receptor levels in C57BL/6J mice. Clozapine and olanzapine, but not haloperidol, induced receptor down-regulation and attenuated PCP-induced locomotor responses. Of the selective 5-HT(2A) antagonists tested, only ketanserin caused significant receptor protein down-regulation, whereas SR46349B up-regulated 5-HT(2A) receptors and potentiated PCP-hyperlocomotion; the other 5-HT(2A) receptor antagonists were without effect. The significance of these findings with respect to atypical antipsychotic drug action is discussed.

Drug-induced valvular heart disease (VHD) is a serious side effect of a few medications, including some that are on the market. Pharmacological studies of VHD-associated medications (e.g., fenfluramine, pergolide, methysergide, and cabergoline) have revealed that they and/or their metabolites are potent 5-hydroxytryptamine(2B) (5-HT(2B)) receptor agonists. We have shown that activation of 5-HT(2B) receptors on human heart valve interstitial cells in vitro induces a proliferative response reminiscent of the fibrosis that typifies VHD. To identify current or future drugs that might induce VHD, we screened approximately 2200 U.S. Food and Drug Administration (FDA)-approved or investigational medications to identify 5-HT(2B) receptor agonists, using calcium-based high-throughput screening. Of these 2200 compounds, 27 were 5-HT(2B) receptor agonists (hits); 14 of these had previously been identified as 5-HT(2B) receptor agonists, including seven bona fide valvulopathogens. Six of the hits (guanfacine, quinidine, xylometazoline, oxymetazoline, fenoldopam, and ropinirole) are approved medications. Twenty-three of the hits were then "functionally profiled" (i.e., assayed in parallel for 5-HT(2B) receptor agonism using multiple readouts to test for functional selectivity). In these assays, the known valvulopathogens were efficacious at concentrations as low as 30 nM, whereas the other compounds were less so. Hierarchical clustering analysis of the pEC(50) data revealed that ropinirole (which is not associated with valvulopathy) was clearly segregated from known valvulopathogens. Taken together, our data demonstrate that patterns of 5-HT(2B) receptor functional selectivity might be useful for identifying compounds likely to induce valvular heart disease.

Ligand-induced regulation of the rat kappa-opioid receptor (rKOR) was investigated in human embryonic kidney 293 cells stably expressing the FLAG-tagged rKOR. Incubation of rKOR cells with naltrexone for 24 h increased the B(max) >3-fold, with no change in the affinity of [(3)H]diprenorphine. Two immunoreactive receptor species were present in cell lysates: naltrexone treatment caused a >3-fold increase in the 52-kDa species while decreasing the level of the 42-kDa species. Dynorphin(1-13), U69,593 [(5alpha,7alpha,8beta)-(+)-N-methyl-N-(7-[1-pyrrolidinyl]-1-oxaspiro[4,5]dec-8-yl)benzeneacetamide], or salvinorin A [2S,4aR,6aR,7R,9S,10aS, 10bR)-9-(acetyloxy)-2-(3-furanyl)dodecahydro-6a,10b-dimethyl-4,10-dioxo-2H-naphtho[2,1c]pyran-7-carboxylic acid methyl ester] treatment did not alter the level of immunoreactive rKOR protein, whereas etorphine, cyclazocine, naloxone, and naloxone methiodide increased the 52-kDa and decreased the 42-kDa rKOR bands. Receptor up-regulation was associated with an increase in the number of cell surface receptors and a 2-fold increase in the E(max) for guanosine 5'-O-(3-[(35)S]thio)triphosphate binding. Glycosidase digestion indicated that the 52- and 42-kDa receptors contained complex and high-mannose N-glycans, respectively, Pulse-chase analysis and glycosidase digestion sensitivities suggested that the 42-kDa rKOR species was a precursor of the 52-kDa species. Naltrexone did not alter rKOR mRNA levels or translational efficiency, and rKOR up-regulation was not inhibited by cycloheximide. Brefeldin A caused accumulation of intracellular rKOR intermediates, and coincubation with naltrexone increased the levels of the brefeldin-induced species significantly. These results suggest that select opioid ligands up-regulate rKOR by enhancing the rate of receptor folding and maturation and by protecting the receptor from degradation, resulting in an increase in the number of rKOR binding sites, immunoreactive protein, and functional receptors.

This study was designed to test the hypothesis that inhibition of agonist-induced delta-receptor down-regulation would block the development of opioid tolerance in a cell-based model. A human embryonic kidney 293 cell line was established that expressed an epitope-tagged delta-opioid receptor (DOR). Treatment of DOR cells with Tyr-d-Ala-Gly-Phe-d-Leu-enkephalin (DADL) resulted in a time-dependent decrease in the B(max) of delta-opioid receptor binding sites and immunoreactive receptor protein. When cells were coincubated with the proteasome inhibitor N-benzyloxycarbonyl-l-leucyl-l-leucyl-l-leucinal (ZLLL) and DADL, the magnitude of the agonist-induced decrease in B(max) and immunoreactive receptor protein was reduced compared with DADL treatment alone. Acute treatment of DOR cells with DADL caused a 3-fold increase in the level of phosphorylated mitogen-activated protein (MAP) kinase. Prior exposure of DOR cells to DADL completely abrogated the agonist-induced activation of MAP kinase. When DOR cells were coincubated with DADL and ZLLL, the proteasome inhibitor prevented the loss of agonist activation of MAP kinase. Acute treatment of DOR cell membranes with DADL stimulated [(35)S]guanosine 5'-3-O-(thio-)triphosphate (GTPgammaS) binding. When DOR cells were preincubated with DADL, the agonist-induced increase in [(35)S]GTPgammaS binding was attenuated. Coincubation of ZLLL and agonist partially prevented the decreased responsiveness to agonist stimulation. The results of this study demonstrated that inhibition of agonist-induced down regulation with a proteasome inhibitor attenuated opioid tolerance in a cellular model, and suggest that coadministration of a proteasome inhibitor with chronic opioid agonist treatment may be useful for limiting opioid tolerance in vivo.

Covering: up to 2004

Hematopoietic regulation is a complex but dynamic process regulated by intercellular and intracellular interactions within the bone marrow (BM) microenvironment. Through neurokinin-1 (NK-1) and NK-2 receptors, peptides (eg, substance P [SP]) encoded by the preprotachykinin-I gene mediate distinct hematopoietic effects. Cytokines, associated with hematopoietic stimulation, and SP regulate the expression of each other in BM mesenchymal and immune cells. Neutral endopeptidase (NEP) uses SP as a substrate to produce SP(1-4), which inhibits the proliferation of matured myeloid progenitor. This study determines whether the degradation of SP to SP(1-4) by endogenous NEP in BM stroma could be a feedback on hematopoietic stimulation by stem cell factor (SCF). SP(1-4) induced the production of transforming growth factor (TGF)–β and tumor necrosis factor–α in BM stroma. TGF–β production accounted for part of the inhibitory effects by SP(1-4) on the proliferation of early (granulocyte-macrophage colony-forming units) and late (long-term culture-initiating cells) hematopoietic progenitors. Enzyme-linked immunosorbent assays and/or protein-chip arrays indicated a timeline change of SP to SP(1-4) in BM stroma stimulated with SCF, which correlated with increase in NEP messenger RNA. Since SP and its fragment, SP(1-4), interact with the same receptor to mediate opposing hematopoietic effects, 2 interactive studies were done to understand the dual responses of NK-1: (1) a 3-dimensional molecular model of NK-1 and SP and (2) screening of a random dodecapeptide library for SP(1-4) interacting sites. The effects of SP(1-4) on hematopoietic progenitors and the timeline change of SP to SP(1-4), together with the 3-dimensional model, provide a partial explanation for the feedback on the stimulatory effects of SCF and SP on hematopoiesis.