Native Rat Research Articles

Identification of UT-A1- and AQP2-interacting proteins in rat inner medullary collecting duct.

The urea channel UT-A1 and the water channel aquaporin-2 (AQP2) mediate vasopressin-regulated transport in the renal inner medullary collecting duct (IMCD). To identify the proteins that interact with UT-A1 and AQP2 in native rat IMCD cells, we carried out chemical cross-linking followed by detergent solubilization, immunoprecipitation, and LC-MS/MS analysis of the immunoprecipitated material. The analyses revealed 133 UT-A1-interacting proteins and 139 AQP2-interacting proteins, each identified in multiple replicates. Fifty-three proteins that were present in both the UT-A1 and the AQP2 interactomes can be considered as mediators of housekeeping interactions, likely common to all plasma membrane proteins. Among proteins unique to the UT-A1 list were those involved in posttranslational modifications: phosphorylation (protein kinases Cdc42bpb, Phkb, Camk2d, and Mtor), ubiquitylation/deubiquitylation (Uba1, Usp9x), and neddylation (Nae1 and Uba3). Among the proteins unique to the AQP2 list were several Rab proteins (Rab1a, Rab2a, Rab5b, Rab5c, Rab7a, Rab11a, Rab11b, Rab14, Rab17) involved in membrane trafficking. UT-A1 was found to interact with UT-A3, although quantitative proteomics revealed that most UT-A1 molecules in the cell are not bound to UT-A3. In vitro incubation of UT-A1 peptides with the protein kinases identified in the UT-A1 interactome revealed that all except Mtor were capable of phosphorylating known sites in UT-A1. Overall, the UT-A1 and AQP2 interactomes provide a snapshot of a dynamic process in which UT-A1 and AQP2 are produced in the rough endoplasmic reticulum, processed through the Golgi apparatus, delivered to endosomes that move into and out of the plasma membrane, and are regulated in the plasma membrane.

A Degraded Fragment of HIV-1 Gp120 in Rat Hepatocytes Forms Fibrils and Enhances HIV-1 Infection

Identification of the host or viral factors that enhance HIV infection is critical for preventing sexual transmission of HIV. Amyloid fibrils derived from human semen, including semen-derived enhancer of virus infection and semenogelins, enhance HIV-1 infection dramatically in vitro. In this study, we reported that a short-degraded peptide fragment 1 (DPF1) derived from native HIV-1 envelope protein gp120-loaded rat hepatocytes, formed fibrils by self-assembly and thus enhanced HIV-1 infection by promoting the binding of HIV-1 to target cells. Furthermore, DPF1-formed fibrils might be used as a crossing seed to accelerate the formation of semen-derived enhancer of virus infection and semenogelin fibrils. It will be helpful to clarify the viral factors that affect HIV-1 infection. DPF1 as an analog of gp120 containing the critical residues for CD4 binding might be useful for designing of HIV vaccines and developing HIV entry inhibitors.

Aryl- and heteroaryl-substituted phenylalanines as AMPA receptor ligands.

A series of racemic unnatural amino acids was rationally designed on the basis of recently published X-ray structures of the GluA2 LBD with bound phenylalanine-based antagonists. Twelve new diaryl- or aryl/heteroaryl-substituted phenylalanine derivatives were synthesized and evaluated in vitro in radioligand binding assays at native rat ionotropic glutamate receptors. The most interesting compound in this series, (RS)-2-amino-3-(3'-hydroxy-5-(1H-pyrazol-4-yl)-[1,1'-biphenyl]-3-yl)propanoic acid 7e, showed the binding affinity of4.6μm for native AMPA receptors and over fourfold lower affinity for kainic acid receptors. Furthermore, 7e was evaluated at recombinant homomeric rat GluA2 and GluA3 receptors. Recently reported X-ray structures 5CBR and 5CBS, representing two distinct antagonist binding modes, were used as templates for molecular docking of the synthesized series. Binding data supported with molecular modeling confirmed that aryl/heteroaryl-substituted phenylalanine analogues effectively bind to AMPA receptors with low micromolar affinity and high selectivity over native NMDA and kainate receptors. These properties make 7e a promising lead for the further development of new AMPA receptor ligands.

Pharmacological characterization and binding modes of novel racemic and optically active phenylalanine-based antagonists of AMPA receptors

In order to map out molecular determinants for the competitive blockade of AMPA receptor subtypes, a series of racemic aryl-substituted phenylalanines was synthesized and pharmacologically characterized in vitro at native rat ionotropic glutamate receptors. Most of the compounds showed micromolar affinity and preference for AMPA receptors. Individual stereoisomers of selected compounds were further evaluated at recombinant homomeric rat GluA2 and GluA3 receptors. The most potent compound, (−)-2-amino-3-(6-chloro-2′,5'-dihydroxy-5-nitro-[1,1'-biphenyl]-3-yl)propanoic acid, the expected R-isomer showing Ki of 1.71 μM at the GluA2 subtype, was found to competitively antagonize GluA2(Q)i receptors in TEVC electrophysiological experiments (Kb = 2.13 μM). Molecular docking experiments allowed us to compare two alternative antagonist binding modes for the synthesized phenylalanines at the GluA2 binding core, showing the direction for further structural modifications.

Biomaterial-Free Three-Dimensional Bioprinting of Cardiac Tissue using Human Induced Pluripotent Stem Cell Derived Cardiomyocytes

We have developed a novel method to deliver stem cells using 3D bioprinted cardiac patches, free of biomaterials. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), fibroblasts (FB) and endothelial cells (EC) were aggregated to create mixed cell spheroids. Cardiac patches were created from spheroids (CM:FB:EC = 70:15:15, 70:0:30, 45:40:15) using a 3D bioprinter. Cardiac patches were analyzed with light and video microscopy, immunohistochemistry, immunofluorescence, cell viability assays and optical electrical mapping. Cardiac tissue patches of all cell ratios beat spontaneously after 3D bioprinting. Patches exhibited ventricular-like action potential waveforms and uniform electrical conduction throughout the patch. Conduction velocities were higher and action potential durations were significantly longer in patches containing a lower percentage of FBs. Immunohistochemistry revealed staining for CM, FB and EC markers, with rudimentary CD31+ blood vessel formation. Immunofluorescence revealed the presence of Cx43, the main cardiac gap junction protein, localized to cell-cell borders. In vivo implantation suggests vascularization of 3D bioprinted cardiac patches with engraftment into native rat myocardium. This constitutes a significant step towards a new generation of stem cell-based treatment for heart failure.

Efficient and Functional Endothelial Repopulation of Whole Lung Organ Scaffolds.

To date, efforts to generate engineered lung tissue capable of long-term function have been limited by incomplete barrier formation between air and blood and by thrombosis of the microvasculature upon exposure of blood to the collagens within the decellularized scaffold. Improved barrier function and resistance to thrombosis both depend upon the recapitulation of a confluent monolayer of functional endothelium throughout the pulmonary vasculature. This manuscript describes novel strategies to increase cell coverage of the vascular surface area, compared to previous reports in our lab and others, and reports robust production of multiple anticoagulant substances that will be key to long-term function in vivo once additional strides are made in improving barrier function. Rat lung microvascular endothelial cells were seeded into decellularized rat lungs by both the pulmonary artery and veins with the use of low-concentration cell suspensions, pulsatile, gravity-driven flow, and supraphysiological vascular pressures. Together, these strategies yielded 72.44 ± 10.52% endothelial cell nuclear coverage of the acellular matrix after 3-4 d of biomimetic bioreactor culture compared to that of the native rat lung. Immunofluorescence, Western blot, and PCR analysis of these lungs indicated robust expression of phenotypic markers such as CD31 and VE-Cadherin after time in culture. Endothelial-seeded lungs had CD31 gene expression of 0.074 ± 0.015 vs 0.021 ± 0.0023 for native lungs, p = 0.025, and VE-Cadherin gene expression of 0.93 ± 0.22 compared to that of the native lung at 0.13 ± 0.02, p = 0.023. Precursors to antithrombotic substances such as tissue plasminogen activator, prostacyclin synthase, and endothelial nitric oxide synthase were expressed at levels equal to or greater than those of the native lung. Engineered lungs reseeded with endothelial cells were implanted orthotopically and contained patent microvascular networks that had gas exchange function during mechanical ventilation on 100% O2 greater than that of decelluarized lungs. Taken together, these data suggest that these engineered constructs could be compatible with long-term function in vivo when utilized in future studies in tandem with improved barrier function.

Controlled gas exchange in whole lung bioreactors.

In cellular, tissue-level or whole organ bioreactors, the level of dissolved oxygen is one of the most important factors requiring control. Hypoxic environments may lead to cellular apoptosis, while hyperoxic environments may lead to cellular damage or dedifferentiation, both resulting in loss of overall tissue function. This manuscript describes the creation, characterization and validation of a bioreactor system that can control oxygen delivery based on real-time metabolic demand of cultured whole lung tissue. A mathematical model describing and predicting gas exchange within the tunable bioreactor system is developed. In addition, the inherent gas exchange properties of the bioreactor and the inherent oxygen consumption rates of native rat lungs are determined, thereby providing a quantitative relationship between system parameters and levels of dissolved oxygen. Finally, the mathematical model is validated during whole lung culture under a range of system parameters. The system presented here provides a quantitative relationship between the concentration of dissolved oxygen, tissue oxygen consumption rates, and controllable system parameters that introduce gasses into the bioreactor. This relationship not only enables the maintenance of constant levels of dissolved oxygen throughout a culture period during which cells are replicating, but also provides noninvasive and real-time estimation of the metabolic and proliferative states of native or engineered lung tissue simply through dissolved oxygen measurements. Copyright © 2017 John Wiley & Sons, Ltd.

Induction of Salivary Gland–Like Cells from Dental Follicle Epithelial Cells

The dental follicle (DF), most often associated with unerupted teeth, is a condensation of ectomesenchymal cells that surrounds the tooth germ in early stages of tooth development. In the present study, we aim to isolate epithelial stem-like cells from the human DF and explore their potential differentiation into salivary gland (SG) cells. We demonstrated the expression of stem cell–related genes in the epithelial components of human DF tissues, and these epithelial progenitor cells could be isolated and ex vivo expanded in a reproducible manner. The human DF-derived epithelial cells possessed clonogenic and sphere-forming capabilities, as well as expressed a panel of epithelial stem cell–related genes, thus conferring stem cell properties (hDF-EpiSCs). When cultured under in vitro 3-dimensional induction conditions, hDF-EpiSCs were capable to differentiate into SG acinar and duct cells. Furthermore, transplantation of hDF-EpiSC–loaded native de-cellularized rat parotid gland scaffolds into the renal capsule of nude mice led to the differentiation of transplanted hDF-EpiSCs into salivary gland–like cells. These findings suggest that hDF-EpiSCs might be a promising source of epithelial stem cells for the development of stem cell–based therapy or bioengineering SG tissues to repair/regenerate SG dysfunction.

Leukotriene receptor expression in mast cells is affected by their agonists

The effects of LTs are mediated by GPCRs: cysLTs interact with CYSLTR1, CYSLTR2, or GPR17, and LTB4 acts via BLT1R or BLT2R. Data relating to the presence of these receptors in mature tissue mast cells are not entirely known. By confocal microscopy with image analyses and flow cytometry, we established that native rat mast cells isolated from peritoneal cavity constitutively express all studied receptors. Moreover, we clearly documented that LTs by themselves can influence their own receptor expression. Low concentrations of LTs induce translocation of LT receptors from cell interior to plasma membrane, which can lead to increased mast cell responsiveness to LT stimulation. High concentrations of LTs cause internalization and, in consequence, reduction in the number of receptors on the cell surface, and it may result in desensitization of mast cells to subsequent LT stimulation. These observations may imply a physiological feedback mechanism regulating mast cell sensitivity to LT activation within tissues.

Asian house rats may facilitate their invasive success through suppressing brown rats in chronic interaction

BackgroundThe Asian house rat (Rattus tanezumi) and the brown rat (Rattus norvegicus) are closely related species and are partially sympatric in southern China. Over the past 20 years, R. tanezumi has significantly expanded northward in China and partially replaced the native brown rat subspecies, R. n. humiliatus. Although invasive species are often more aggressive than native species, we did not observe interspecific physical aggression between R. tanezumi and R. n. humiliatus. Here, we focused on whether or not R. tanezumi was superior to R. n. humiliatus in terms of nonphysical competition, which is primarily mediated by chemical signals.ResultsWe performed two laboratory experiments to test different paradigms in domesticated R. tanezumi and R. n. humiliatus. In Experiment 1, we caged adult male rats of each species for 2 months in heterospecific or conspecific pairs, partitioned by perforated galvanized iron sheets, allowing exchange of chemical stimuli and ultrasonic vocalization. The sexual attractiveness of male urine odor showed a tendency (marginal significance) to increase in R. tanezumi caged with R. n. humiliatus, compared with those in conspecific pairs. Hippocampal glucocorticoid receptor (GR) and brain-derived nutrition factor (BDNF) mRNA were upregulated in R. n. humiliatus and R. tanezumi, respectively, when the rats were caged in heterospecific pairs. In Experiment 2, we kept juvenile male rats in individual cages in rooms with either the same or the different species for 2 months, allowing chemical interaction. The sexual attractiveness of male urine was significantly enhanced in R. tanezumi, but reduced in R. n. humiliatus by heterospecific cues and mRNA expression of hippocampal GR and BDNF were upregulated by heterospecific cues in R. n. humiliatus and R. tanezumi, respectively. Although not identical, the results from Experiments 1 and 2 were generally consistent.ConclusionsThe results of both experiments indicate that nonphysical/chronic interspecific stimuli, particularly scent signals, between R. n. humiliatus and R. tanezumi may negatively affect R. n. humiliatus and positively affect R. tanezumi. We infer that chronic interspecific interactions may have contributed to the invasion of R. tanezumi into the range of R. n. humiliatus in natural habitats.

Identification of β‐Catenin Interacting Proteins in the Nuclei of Native Rat Renal Inner Medullary Collecting Duct Cells

The gene encoding the aquaporin‐2 water channel (AQP2) is regulated transcriptionally in response to vasopressin. Vasopressin increases phosphorylation of many proteins in collecting duct cells including AQP2 and β‐catenin (at Serine‐552), a multifunctional protein that can function as a transcriptional co‐regulator in the nucleus. β‐catenin phosphorylation is associated with translocation of β‐catenin from the cytoplasm to the nucleus of collecting duct cells where it may bind to transcription factors and other regulatory proteins. The purpose of this study was to identify β‐catenin interacting proteins in the nuclei of inner medullary collecting duct (IMCD) cells isolated from rat kidneys using chromatin immunoprecipitation coupled to mass spectrometry (ChIP‐MS). Samples were analyzed from rat IMCD suspensions treated either with the vasopressin analog dDAVP or its vehicle for 30 minutes in vitro. After crosslinking and nuclear isolation, chromatin immunoprecipitation was performed using a well characterized β‐catenin antibody (or with pre‐immune IgG). The co‐immunoprecipitated proteins were identified by protein mass spectrometry (n=4 for both dDAVP and control). We reproducibly identified 43 β‐catenin binding proteins present with β‐catenin antibody but not pre‐immune IgG, which included a number of known β‐catenin binding partners as well as many novel interacting proteins. There were no sequence‐specific transcription factors, but there were multiple proteins known to be involved in transcriptional regulation (Jup, Tdrd3, Lgr4, Cdh1, Cenpj, Taf1, and several histones). Binding of some proteins to β‐catenin changed in response to dDAVP (Minimum Bayes' Factor < 0.01) (Plec [down], Cdh1 [up], Krt4 [up], Lrba [up], Sptbn2 [up], and Tenm3 [up]). These findings provide key information necessary for modeling the transcriptional response to vasopressin. [JH supported by APS Undergraduate Summer Research Fellowship Program.]Support or Funding InformationThe work was supported by the APS Undergraduate Summer Research Fellowship and NHLBI Intramural Research Program (project ZIA‐HL001285 and ZIA‐HL006129, M.A.K.).

Identification of β-catenin-interacting proteins in nuclear fractions of native rat collecting duct cells.

The gene encoding the aquaporin-2 water channel is regulated transcriptionally in response to vasopressin. In the renal collecting duct, vasopressin stimulates the nuclear translocation and phosphorylation (at Ser552) of β-catenin, a multifunctional protein that acts as a transcriptional coregulator in the nucleus. The purpose of this study was to identify β-catenin-interacting proteins that might be involved in transcriptional regulation in rat inner medullary collecting duct (IMCD) cells, using experimental and computational approaches. We used a standard chromatin immunoprecipitation procedure coupled to mass spectrometry (ChIP-MS) in a nuclear fraction isolated from rat IMCD suspensions. Over four biological replicates, we reproducibly identified 43 β-catenin-binding proteins, including several known β-catenin-binding partners as well as novel interacting proteins. Multiple proteins involved in transcriptional regulation were identified (Taf1, Jup, Tdrd3, Cdh1, Cenpj, and several histones). Many of the identified β-catenin-binding partners were found in prior studies to translocate to the nucleus in response to vasopressin. There was only one DNA-binding transcription factor (TF), specifically Taf1, part of the RNA-polymerase II preinitiation complex. To identify sequence-specific TFs that might interact with β-catenin, Bayes' theorem was used to integrate data from several information sources. The analysis identified several TFs with potential binding sites in the Aqp2 gene promoter that could interact with β-catenin in the regulation of Aqp2 gene transcription, specifically Jun, Junb, Jund, Atf1, Atf2, Mef2d, Usf1, Max, Pou2f1, and Rxra. The findings provide information necessary for modeling the transcriptional response to vasopressin.

Generation, Endothelialization, and Microsurgical Suture Anastomosis of Strong 1-mm-Diameter Collagen Tubes.

Tissue-engineered vascular grafts that are based on reconstituted extracellular matrices have been plagued by weak mechanical strength that prevents handling or anastomosis to native vessels. In this study, we devise a method for making dense, suturable collagen tubular constructs of diameter ≤1 mm for potential microsurgical applications, by dehydrating tubes of native rat tail type I collagen and crosslinking them with 20 mM genipin. Crosslinked dense collagen tubes with 1 mm inner diameter yielded ultimate tensile strength of 342 ± 15 gF and burst pressure of 1313 ± 156 mm Hg, comparable to the strength of a rat femoral artery, and supported endothelial cell adhesion and growth. End-to-end anastomosis of 0.5-mm-diameter tubes to explanted arteries displayed anastomotic strength of 82 ± 21 gF, which is sufficient for surgical applications. In vivo implantation of cell-free tubes as interpositional grafts in the rat femoral circulation yielded stable anastomosis with blood flow for 20 min. Seeded dense collagen tubes represent a promising alternative to venous graft that can potentially be used to bridge between short artery stubs in replantation surgeries.

Biomechanical properties of an implanted engineered tubular gut-sphincter complex.

Neuromuscular diseases of the gut alter the normal motility patterns. Although surgical intervention remains the standard treatment, preservation of the sphincter attached to the rest of the gut is challenging. The present study aimed to evaluate a bioengineered gut-sphincter complex following its subcutaneous implantation for 4weeks in rats. Engineered innervated human smooth muscle sheets and innervated human sphincters with a predefined alignment were placed around tubular scaffolds to create a gut-sphincter complex. The engineered complex was subcutaneously implanted in the abdomen of the rats for 4weeks. The implanted tissues were vascularized. In vivo manometry revealed luminal pressure at the gut and the sphincter zone. Tensile strength, elongation at break and Young's modulus of the engineered complexes were similar to those of native rat intestine. Histological and immunofluorescence assays showed maintenance of smooth muscle circular alignment in the engineered tissue, maintenance of smooth muscle contractile phenotype and innervation of the smooth muscle. Electrical field stimulation induced relaxation of the smooth muscle of both the sphincter and the gut parts. Relaxation was partly inhibited by nitric oxide inhibitor indicating nitrergic contribution to relaxation. The present study has demonstrated for the first time a successfully developed and subcutaneously implanted a tubular human-derived gut-sphincter complex. The sphincteric part of Tubular Gut-Sphincter Complex (TGSC) maintained the basal tone characteristic of a native sphincter. The gut part also maintained its specific neuromuscular characteristics. The results of this study provide a promising therapeutic approach to restore gut continuity and motility. Copyright © 2016 John Wiley & Sons, Ltd.

The novel H2 S donor 4-carboxy-phenyl isothiocyanate inhibits mast cell degranulation and renin release by decreasing intracellular calcium.

Hydrogen sulfide (H2 S) modulates many pathophysiological processes, including inflammation and allergic reactions, in which mast cells act as major effector cells. IgE receptor (FcεRI) cross linking leads to an increase in intracellular calcium ([Ca+2 ]i ), a critical step in mast cell degranulation. The aim of this study was to investigate the role of H2 S in [Ca+2 ]i -dependent mast cell activation. We investigated the effects of H2 S, either endogenously produced or released by the slow H2 S donor 4-carboxy-phenyl isothiocyanate (PhNCS-COOH), on antigenic- and non-antigenic degranulation of native murine mast cells, and human and rat (RBL-2H3) mast cell lines. We measured the release of specific mast cell degranulation markers (β-hexosaminidase and renin), as well as changes in [Ca+2 ]i and phosphorylation of proteins downstream of FcεRI activation. Endogenously produced H2 S inhibited antigen-induced degranulation in RBL-2H3. Similarly, H2 S released by PhNCS-COOH (10-300μM) reduced, in a concentration-dependent manner, antigenic and non-antigenic degranulation and renin release in all mast cell types. Notably, PhNCS-COOH also prevented in a concentration-dependent mode the increase in [Ca+2 ]i elicited by Ca+2 ionophore, thapsigargin and FcεRI activation. Moreover, PhNCS-COOH attenuated the phosphorylation of Syk, cPLA-2 and PLCγ1 in antigen-stimulated RBL-2H3 cells. Collectively, our results demonstrate that, by attenuating the phosphorylation of proteins downstream of FcεRI cross-linking on mast cells, H2 S diminishes [Ca+2 ]i availability and thus mast cell degranulation and renin release. These findings suggest that PhNCS-COOH could be a strategic therapeutic tool in mast cell-mediated allergic conditions.

Design, synthesis and structure–activity relationships of novel phenylalanine-based amino acids as kainate receptors ligands

A new series of carboxyaryl-substituted phenylalanines was designed, synthesized and pharmacologically characterized in vitro at native rat ionotropic glutamate receptors as well as at cloned homomeric kainate receptors GluK1–GluK3. Among them, six compounds bound to GluK1 receptor subtypes with reasonable affinity (Ki values in the range of 4.9–7.5μM). A structure–activity relationship (SAR) for the obtained series, focused mainly on the pharmacological effect of structural modifications in the 4- and 5-position of the phenylalanine ring, was established. To illustrate the results, molecular docking of the synthesized series to the X-ray structure of GluK1 ligand binding core was performed. The influence of individual substituents at the phenylalanine ring for both the affinity and selectivity at AMPA, GluK1 and GluK3 receptors was analyzed, giving directions for future studies.

Opposite effects of acute and chronic amphetamine on Nurr1 and NF-κB p65 in the rat ventral tegmental area

Dopamine neurons are overstimulated by drugs of abuse and suffer molecular alterations that lead to addiction behavior. Nurr1 is a transcription factor crucial for dopamine neurons survival and dopamine production, activating the transcription of key genes like tyrosine hydroxylase (TH). Interestingly, nuclear factor-kappa B (NF-κB) has emerged as a new Nurr1 partner in response to inflammatory stimulus. In this study we evaluated the effects of single and repeated amphetamine administration in the expression of Nurr1 and the NF-κB p65 subunit in the rat ventral tegmental area (VTA). We found that acute amphetamine treatment increased Nurr1, p65 and TH protein levels in the VTA. On the other hand, chronic amphetamine treatment decreased Nurr1 and p65 protein levels, but TH was unchanged. Mammalian reporter assays in cell lines showed that p65 represses Nurr1 transcriptional activity in an artificial promoter driven by Nurr1 response elements and in the native rat TH promoter. These results indicate that Nurr1 and NF-κB p65 factors are involved in the adaptive response of dopamine neurons to psychostimulants and that both transcription factors could be regulating Nurr1-dependent transactivation in the VTA.

Discovery of α-Substituted Imidazole-4-acetic Acid Analogues as a Novel Class of ρ1 γ-Aminobutyric Acid Type A Receptor Antagonists with Effect on Retinal Vascular Tone.

The ρ-containing γ-aminobutyric acid type A receptors (GABAA Rs) play an important role in controlling visual signaling. Therefore, ligands that selectively target these GABAA Rs are of interest. In this study, we demonstrate that the partial GABAA R agonist imidazole-4-acetic acid (IAA) is able to penetrate the blood-brain barrier in vivo; we prepared a series of α- and N-alkylated, as well as bicyclic analogues of IAA to explore the structure-activity relationship of this scaffold focusing on the acetic acid side chain of IAA. The compounds were prepared via IAA from l-histidine by an efficient minimal-step synthesis, and their pharmacological properties were characterized at native rat GABAA Rs in a [3 H]muscimol binding assay and at recombinant human α1 β2 γ2S and ρ1 GABAA Rs using the FLIPR™ membrane potential assay. The (+)-α-methyl- and α-cyclopropyl-substituted IAA analogues ((+)-6 a and 6 c, respectively) were identified as fairly potent antagonists of the ρ1 GABAA R that also displayed significant selectivity for this receptor over the α1 β2 γ2S GABAA R. Both 6 a and 6 c were shown to inhibit GABA-induced relaxation of retinal arterioles from porcine eyes.

The Novel, Nicotinic Alpha7 Receptor Partial Agonist, BMS-933043, Improves Cognition and Sensory Processing in Preclinical Models of Schizophrenia.

The development of alpha7 nicotinic acetylcholine receptor agonists is considered a promising approach for the treatment of cognitive symptoms in schizophrenia patients. In the present studies we characterized the novel agent, (2R)-N-(6-(1H-imidazol-1-yl)-4-pyrimidinyl)-4'H-spiro[4-azabicyclo[2.2.2]octane-2,5'-[1,3]oxazol]-2'-amine (BMS-933043), in vitro and in rodent models of schizophrenia-like deficits in cognition and sensory processing. BMS-933043 showed potent binding affinity to native rat (Ki = 3.3 nM) and recombinant human alpha7 nicotinic acetylcholine receptors (Ki = 8.1 nM) and agonist activity in a calcium fluorescence assay (EC50 = 23.4 nM) and whole cell voltage clamp electrophysiology (EC50 = 0.14 micromolar (rat) and 0.29 micromolar (human)). BMS-933043 exhibited a partial agonist profile relative to acetylcholine; the relative efficacy for net charge crossing the cell membrane was 67% and 78% at rat and human alpha7 nicotinic acetylcholine receptors respectively. BMS-933043 showed no agonist or antagonist activity at other nicotinic acetylcholine receptor subtypes and was at least 300 fold weaker at binding to and antagonizing human 5-HT3A receptors (Ki = 2,451 nM; IC50 = 8,066 nM). BMS-933043 treatment i) improved 24 hour novel object recognition memory in mice (0.1–10 mg/kg, sc), ii) reversed MK-801-induced deficits in Y maze performance in mice (1–10 mg/kg, sc) and set shift performance in rats (1–10 mg/kg, po) and iii) reduced the number of trials required to complete the extradimensional shift discrimination in neonatal PCP treated rats performing the intra-dimensional/extradimensional set shifting task (0.1–3 mg/kg, po). BMS-933043 also improved auditory gating (0.56–3 mg/kg, sc) and mismatch negativity (0.03–3 mg/kg, sc) in rats treated with S(+)ketamine or neonatal phencyclidine respectively. Given this favorable preclinical profile BMS-933043 was selected for further development to support clinical evaluation in humans.

A pharmacological profile of the high-affinity GluK5 kainate receptor

Mouse GluK5 was expressed in Sf9 insect cells and radiolabelled with [3H]-kainate in receptor binding assays (Kd=6.9nM). Western immunoblotting indicated an Sf9 GluK5 band doublet corresponding to the glycosylated (128kDa) and deglycosylated (111kDa) protein, which was identical to the band pattern of native rat brain GluK5. A pharmacological profile of the high-affinity kainate receptor GluK5 is described which is distinct from the profiles of other kainate receptors (GluK1-3). The 27 tested ligands generally show a preferential affinity to GluK1 over GluK5, the exceptions being: dihydrokainate, (S)-5-fluorowillardiine, (S)-glutamate and quisqualate, where the affinity is similar at GluK1 and GluK5. In contrast, quisqualate shows 40-fold higher affinity at GluK5 over GluK3 whereas (S)-1-(2′-amino-2′-caboxyethyl)thienol[3,4-d]pyrimidin-2,4-dione (NF1231), (RS)-2-amino-3-(5-tert-butyl-3-hydroxyisoxazol-4-yl)propionate (ATPA), dihydrokainate and (2S,4R)-4-methyl-glutamate (SYM2081) have higher affinity at GluK3 compared to GluK5. Since some studies have indicated that GluK5 is associated with various diseases in the central nervous system (e.g. schizophrenia, temporal lobe epilepsy, bipolar disorder), selective GluK5 ligands could have therapeutic potential. The distinct pharmacological profile of GluK5 suggests that it would be possible to design ligands with selectivity towards GluK5.