Chemical ablation of gastric inhibitory polypeptide receptor action by daily (Pro3) GIP administration improves glucose tolerance and ameliorates insulin resistance and abnormalities of islet structure in obesity-diabetes.

Glucose-dependent insulinotropic polypeptide (gastric inhibitory polypeptide [GIP]) is an important incretin hormone secreted by endocrine K-cells in response to nutrient ingestion. In this study, we investigated the effects of chemical ablation of GIP receptor (GIP-R) action on aspects of obesity-related diabetes using a stable and specific GIP-R antagonist, (Pro3)GIP. Young adult ob/ob mice received once-daily intraperitoneal injections of saline vehicle or (Pro3)GIP over an 11-day period. Nonfasting plasma glucose levels and the overall glycemic excursion (area under the curve) to a glucose load were significantly reduced (1.6-fold; P <0.05) in (Pro3)GIP-treated mice compared with controls. GIP-R ablation also significantly lowered overall plasma glucose (1.4-fold; P <0.05) and insulin (1.5-fold; P <0.05) responses to feeding. These changes were associated with significantly enhanced (1.6-fold; P <0.05) insulin sensitivity in the (Pro3)GIP-treated group. Daily injection of (Pro3)GIP reduced pancreatic insulin content (1.3-fold; P <0.05) and partially corrected the obesity-related islet hypertrophy and ß-cell hyperplasia of ob/ob mice. These comprehensive beneficial effects of (Pro3)GIP were reversed 9 days after cessation of treatment and were independent of food intake and body weight, which were unchanged. These studies highlight a role for GIP in obesity-related glucose intolerance and emphasize the potential of specific GIP-R antagonists as a new class of drugs for the alleviation of insulin resistance and treatment of type 2 diabetes.

Differential effects of an anti-oxidant intervention on cardiomyocyte expression of adrenomedullin and intermedin and their receptor components in chronic nitric oxide deficiency

Background: Chronic inhibition of nitric oxide (NO) synthesis is associated with hypertension, myocardial oxidative stress and hypertrophic remodeling. Up-regulation of the cardiomyocyte adrenomedullin (AM) / intermedin (IMD) receptor signaling cascade is also apparent in NO-deficient cardiomyocytes: augmented expression of AM and receptor activity modifying proteins RAMP2 and RAMP3 is prevented by blood pressure normalization while that of RAMP1 and intermedin (IMD) is not, indicating that the latter is regulated by a pressure-independent mechanism. Aims: to verify the ability of an anti-oxidant intervention to normalize cardiomyocyte oxidant status and to investigate the influence of such an intervention on expression of AM, IMD and their receptor components in NO-deficient cardiomyocytes. Methods: NO synthesis inhibitor, NG-nitro-L-arginine methyl ester (L-NAME, 35mg/kg/day) was given to rats for 8 weeks, with/without con-current administration of antioxidants (Vitamin C (25mg/kg/day) and Tempol (25mg/kg/day)). Results: In left ventricular cardiomyocytes isolated from L-NAME treated rats, increased oxidative stress was indicated by augmented (3.6 fold) membrane protein oxidation, enhanced expression of catalytic and regulatory subunits of pro-oxidant NADPH oxidases (NOX1, NOX2) and compensatory increases in expression of anti-oxidant glutathione peroxidase and Cu/Zn superoxide dismutases (SOD1, SOD3). Vitamin C plus Tempol did not reduce systolic blood pressure but normalized augmented plasma levels of IMD, but not of AM, and in cardiomyocytes: (i) abolished increased membrane protein oxidation; (ii) normalized augmented expression of prepro-IMD and RAMP1, but not prepro-AM, RAMP2 and RAMP3; (iii) attenuated (by 42%) increased width and normalized expression of hypertrophic markers, skeletal-�-actin and prepro-endothelin-1 similarly to blood pressure normalization but in contrast to blood pressure normalization did not attenuate augmented brain natriuretic peptide (BNP) expression. Conclusion: normalization specifically of augmented IMD/RAMP1 expression in NO-deficient cardiomyocytes by antioxidant intervention in the absence of blood pressure reduction indicates that these genes are likely to be induced directly by myocardial oxidative stress. Although oxidative stress contributed to cardiomyocyte hypertrophy, induction of IMD and RAMP1 is unlikely to be secondary to cardiomyocyte hypertrophy.

Interleukin-8 signaling promotes androgen-independent proliferation of prostate cancer cells via induction of androgen receptor expression and activation

The aim of our study was to assess the importance of the CXC chemokine and interleukin (IL)-8 in promoting the transition of prostate cancer (CaP) to the androgen-independent state. Stimulation of the androgen-dependent cell lines, LNCaP and 22Rv1, with exogenous recombinant human interleukin-8 (rh-IL-8) increased androgen receptor (AR) gene expression at the messenger RNA (mRNA) and protein level, assessed by quantitative polymerase chain reaction and immunoblotting, respectively. Using an androgen response element-luciferase construct, we demonstrated that rh-IL-8 treatment also resulted in increased AR transcriptional activity in both these cell lines, and a subsequent upregulation of prostate-specific antigen and cyclin-dependent kinase 2 mRNA transcript levels in LNCaP cells. Blockade of CXC chemokine receptor-2 signaling using a small molecule antagonist (AZ10397767) attenuated the IL-8-induced increases in AR expression and transcriptional activity. Furthermore, in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, coadministration of AZ10397767 reduced the viability of LNCaP and 22Rv1 cells exposed to bicalutamide. Our data show that IL-8 signaling increases AR expression and promotes ligand-independent activation of this receptor in two androgen-dependent cell lines, describing two mechanisms by which this chemokine may assist in promoting the transition of CaP to the androgen-independent state. In addition, our data show that IL-8-promoted regulation of the AR attenuates the effectiveness of the AR antagonist bicalutamide in reducing CaP cell viability.

Chemotherapy-Induced CXC-Chemokine/CXC-Chemokine Receptor Signaling in Metastatic Prostate Cancer Cells Confers Resistance to Oxaliplatin through Potentiation of Nuclear Factor-κB Transcription and Evasion of Apoptosis

Constitutive activation of nuclear factor (NF)-kappa B is linked with the intrinsic resistance of androgen-independent prostate cancer (AIPC) to cytotoxic chemotherapy. Interleukin-8 (CXCL8) is a transcriptional target of NF-kappa B whose expression is elevated in AIPC. This study sought to determine the significance of CXCL8 signaling in regulating the response of AIPC cells to oxaliplatin, a drug whose activity is reportedly sensitive to NF-kappa B activity. Administration of oxaliplatin to PC3 and DU145 cells increased NF-kappa B activity, promoting antiapoptotic gene transcription. In addition, oxaliplatin increased the transcription and secretion of CXCL8 and the related CXC-chemokine CXCL1 and increased the transcription and expression of CXC-chemokine receptors, especially CXC-chemokine receptor (CXCR) 2, which transduces the biological effects of CXCL8 and CXCL1. Stimulation of AIPC cells with CXCL8 potentiated NF-kappa B activation in AIPC cells, increasing the transcription and expression of NF-kappa B-regulated antiapoptotic genes of the Bcl-2 and IAP families. Coadministration of a CXCR2-selective antagonist, AZ10397767 (Bioorg Med Chem Lett 18:798-803, 2008), attenuated oxaliplatin-induced NF-kappa B activation, increased oxaliplatin cytotoxicity, and potentiated oxaliplatin-induced apoptosis in AIPC cells. Pharmacological inhibition of NF-kappa B or RNA interference-mediated suppression of Bcl-2 and survivin was also shown to sensitize AIPC cells to oxaliplatin. Our results further support NF-kappa B activity as an important determinant of cancer cell sensitivity to oxaliplatin and identify the induction of autocrine CXCR2 signaling as a novel mode of resistance to this drug.

Combined inhibition of FLIP and XIAP induces Bax-independent apoptosis in type II colorectal cancer cells.

Death receptors can directly (type I cells) or indirectly induce apoptosis by activating mitochondrial-regulated apoptosis (type II cells). The level of caspase 8 activation is thought to determine whether a cell is type I or II, with type II cells less efficient at activating this caspase following death receptor activation. FLICE-inhibitory protein (FLIP) blocks death receptor-mediated apoptosis by inhibiting caspase 8 activation; therefore, we assessed whether silencing FLIP could convert type II cells into type I. FLIP silencing-induced caspase 8 activation in Bax wild-type and null HCT116 colorectal cancer cells; however, complete caspase 3 processing and apoptosis were only observed in Bax wild-type cells. Bax-null cells were also more resistant to chemotherapy and tumor necrosis factor-related apoptosis inducing ligand and, unlike the Bax wild-type cells, were not sensitized to these agents by FLIP silencing. Further analyses indicated that release of second mitochondrial activator of caspases from mitochondria and subsequent inhibition of X-linked inhibitor of apoptosis protein (XIAP) was required to induce full caspase 3 processing and apoptosis following FLIP silencing. These results indicate that silencing FLIP does not necessarily bypass the requirement for mitochondrial involvement in type II cells. Furthermore, targeting FLIP and XIAP may represent a therapeutic strategy for the treatment of colorectal tumors with defects in mitochondrial-regulated apoptosis.

Prognostic Significance of TRAIL Signaling Molecules in Stage II and III Colorectal Cancer

Purpose: We previously found that cellular FLICE-inhibitory protein (c-FLIP), caspase 8, and tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) receptor 2 (DR5) are major regulators of cell viability and chemotherapy-induced apoptosis in colorectal cancer. In this study, we determined the prognostic significance of c-FLIP, caspase 8, TRAIL and DR5 expression in tissues from patients with stage II and III colorectal cancer.

Experimental Design: Tissue microarrays were constructed from matched normal and tumor tissue derived from patients (n = 253) enrolled in a phase III trial of adjuvant 5-fluorouracil–based chemotherapy versus postoperative observation alone. TRAIL, DR5, caspase 8, and c-FLIP expression levels were determined by immunohistochemistry.

Results: Colorectal tumors displayed significantly higher expression levels of c-FLIP (P < 0.001), caspase 8 (P = 0.01), and DR5 (P < 0.001), but lower levels of TRAIL (P < 0.001) compared with matched normal tissue. In univariate analysis, higher TRAIL expression in the tumor was associated with worse overall survival (P = 0.026), with a trend to decreased relapse-free survival (RFS; P = 0.06), and higher tumor c-FLIP expression was associated with a significantly decreased RFS (P = 0.015). Using multivariate predictive modeling for RFS in all patients and including all biomarkers, age, treatment, and stage, we found that the model was significant when the mean tumor c-FLIP expression score and disease stage were included (P < 0.001). As regards overall survival, the overall model was predictive when both TRAIL expression and disease stage were included (P < 0.001).

Conclusions: High c-FLIP and TRAIL expression may be independent adverse prognostic markers in stage II and III colorectal cancer and might identify patients most at risk of relapse.

Cell-collagen interactions: the use of peptide Toolkits to investigate collagen-receptor interactions

Fibrillar collagens provide the most fundamental platform in the vertebrate organism for the attachment of cells and matrix molecules. we have identified specific sites in collagens to which cells can attach, either directly or through protein intermediaries. Using Toolkits of triple-helical peptides, each peptide comprising 27 residues of collagen primary sequence and overlapping with its neighbours by nine amino acids, we have mapped the binding of receptors and other proteins on to collagens II or III. Integrin alpha 2 beta 1 binds to several GXX'GER motifs within the collagens, the affinities of which differ sufficiently to control cell adhesion and migration independently of the cellular regulation of the integrin. The platelet receptor, Gp (glycoprotein) VI binds well to GPO (where 0 is hydroxyproline)-containing model peptides, but to very few Toolkit peptides, suggesting that sequence in addition to GPO triplets is important in defining GpVI binding. The Toolkits have been applied to the plasma protein vWF (von Willebrand factor), which binds to only a single sequence, identified by truncation and amino acid substitution within Toolkit peptides, as GXRGQOGVMGFO in collagens II and III. Intriguingly, the receptor tyrosine kinase, DDR2 (discoidin domain receptor 2) recognizes three sites in collagen II, including its vWF-binding site, although the amino acids that support the interaction differ slightly within this motif. Furthermore, the secreted protein BM-40 (basement membrane protein 40) also binds well to this same region. Thus the availability of extracellular collagen-binding proteins may be important in regulating and facilitating direct collagen-receptor interaction.

Identification of ML204, a novel potent antagonist that selectively modulates native TRPC4/C5 ion channels.

Transient receptor potential canonical (TRPC) channels are Ca(2+)-permeable nonselective cation channels implicated in diverse physiological functions, including smooth muscle contractility and synaptic transmission. However, lack of potent selective pharmacological inhibitors for TRPC channels has limited delineation of the roles of these channels in physiological systems. Here we report the identification and characterization of ML204 as a novel, potent, and selective TRPC4 channel inhibitor. A high throughput fluorescent screen of 305,000 compounds of the Molecular Libraries Small Molecule Repository was performed for inhibitors that blocked intracellular Ca(2+) rise in response to stimulation of mouse TRPC4ß by µ-opioid receptors. ML204 inhibited TRPC4ß-mediated intracellular Ca(2+) rise with an IC(50) value of 0.96 µm and exhibited 19-fold selectivity against muscarinic receptor-coupled TRPC6 channel activation. In whole-cell patch clamp recordings, ML204 blocked TRPC4ß currents activated through either µ-opioid receptor stimulation or intracellular dialysis of guanosine 5'-3-O-(thio)triphosphate (GTP?S), suggesting a direct interaction of ML204 with TRPC4 channels rather than any interference with the signal transduction pathways. Selectivity studies showed no appreciable block by 10-20 µm ML204 of TRPV1, TRPV3, TRPA1, and TRPM8, as well as KCNQ2 and native voltage-gated sodium, potassium, and calcium channels in mouse dorsal root ganglion neurons. In isolated guinea pig ileal myocytes, ML204 blocked muscarinic cation currents activated by bath application of carbachol or intracellular infusion of GTP?S, demonstrating its effectiveness on native TRPC4 currents. Therefore, ML204 represents an excellent novel tool for investigation of TRPC4 channel function and may facilitate the development of therapeutics targeted to TRPC4.

Context-Dependent Pharmacology Exhibited by Negative Allosteric Modulators of Metabotropic Glutamate Receptor 7

Phenotypic studies of mice lacking metabotropic glutamate receptor subtype 7 (mGluR7) suggest that antagonists of this receptor may be promising for the treatment of central nervous system disorders such as anxiety and depression. Suzuki et al. (J Pharmacol Exp Ther 323: 147-156, 2007) recently reported the in vitro characterization of a novel mGluR7 antagonist called 6-(4-methoxyphenyl)-5-methyl-3-(4-pyridinyl)-isoxazolo[4,5-c]pyridin-4(5H)-one (MMPIP), which noncompetitively inhibited the activity of orthosteric and allosteric agonists at mGluR7. We describe that MMPIP acts as a noncompetitive antagonist in calcium mobilization assays in cells coexpressing mGluR7 and the promiscuous G protein G alpha(15). Assessment of the activity of a small library of MMPIP-derived compounds using this assay reveals that, despite similar potencies, compounds exhibit differences in negative co-operativity for agonist-mediated calcium mobilization. Examination of the inhibitory activity of MMPIP and analogs using endogenous G(i/o)-coupled assay readouts indicates that the pharmacology of these ligands seems to be context-dependent, and MMPIP exhibits differences in negative cooperativity in certain cellular backgrounds. Electrophysiological studies reveal that, in contrast to the orthosteric antagonist (2S)-2-amino-2-[(1S,2S)-2-carboxyclycloprop-1-yl]-3-(xanth-9-yl) propanoic acid (LY341495), MMPIP is unable to block agonist-mediated responses at the Schaffer collateral-CA1 synapse, a location at which neurotransmission has been shown to be modulated by mGluR7 activity. Thus, MMPIP and related compounds differentially inhibit coupling of mGluR7 in different cellular backgrounds and may not antagonize the coupling of this receptor to native G(i/o) signaling pathways in all cellular contexts. The pharmacology of this compound represents a striking example of the potential for context-dependent blockade of receptor responses by negative allosteric modulators.

Comparison of Dynamics of Extracellular Accesses to the β(1) and β(2) Adrenoceptors Binding Sites Uncovers the Potential of Kinetic Basis of Antagonist Selectivity.

From the molecular mechanism of antagonist unbinding in the ß(1) and ß(2) adrenoceptors investigated by steered molecular dynamics, we attempt to provide further possibilities of ligand subtype and subspecies selectivity. We have simulated unbinding of ß(1) -selective Esmolol and ß(2) -selective ICI-118551 from both receptors to the extracellular environment and found distinct molecular features of unbinding. By calculating work profiles, we show different preference in antagonist unbinding pathways between the receptors, in particular, perpendicular to the membrane pathway is favourable in the ß(1) adrenoceptor, whereas the lateral pathway involving helices 5, 6 and 7 is preferable in the ß(2) adrenoceptor. The estimated free energy change of unbinding based on the preferable pathway correlates with the experimental ligand selectivity. We then show that the non-conserved K347 (6.58) appears to facilitate in guiding Esmolol to the extracellular surface via hydrogen bonds in the ß(1) adrenoceptor. In contrast, hydrophobic and aromatic interactions dominate in driving ICI-118551 through the easiest pathway in the ß(2) adrenoceptor. We show how our study can stimulate design of selective antagonists and discuss other possible molecular reasons of ligand selectivity, involving sequential binding of agonists and glycosylation of the receptor extracellular surface. © 2012 John Wiley & Sons A/S.

P2Y(1) antagonists: Combining receptor-based modeling and QSAR for a quantitative prediction of the biological activity based on consensus scoring

P2Y(1) is an ADP-activated G protein-coupled receptor (GPCR). Its antagonists impede platelet aggregation in vivo and are potential antithrombotic agents. Combining ligand and structure-based modeling we generated a consensus model (LIST-CM) correlating antagonist structures with their potencies. We docked 45 antagonists into our rhodopsin-based human P2Y(1) homology model and calculated docking scores and free binding energies with the Linear Interaction Energy (LIE) method in continuum-solvent. The resulting alignment was also used to build QSAR based on CoMFA, CoMSIA, and molecular descriptors. To benefit from the strength of each technique and compensate for their limitations, we generated our LIST-CM with a PLS regression based on the predictions of each methodology. A test set featuring untested substituents was synthesized and assayed in inhibition of 2-MeSADP-stimulated PLC activity and in radioligand binding. LIST-CM outperformed internal and external predictivity of any individual model to predict accurately the potency of 75% of the test set.

The agonism and synergistic potentiation of weak partial agonists by triethylamine in alpha(1)-adrenergic receptor activation: Evidence for a salt bridge as the initiating process

alpha(1)-adrenergic receptor (AR) activation is thought to be initiated by disruption of a constraining interhelical salt bridge (Porter et al., 1996). Disruption of this salt bridge is achieved through a competition for the aspartic acid residue in transmembrane domain three by the protonated amine of the endogenous ligand norepinephrine and a lysine residue in transmembrane domain seven. To further test this hypothesis, we investigated the possibility that a simple amine could mimic an important functional group of the endogenous ligand and break this alpha(1)-AR ionic constraint leading to agonism. Triethylamine (TEA) was able to generate concentration-dependent increases of soluble inositol phosphates in COS-1 cells transiently transfected with the hamster alpha(1b)-AR and in Rat-1 fibroblasts stably transfected with the human alpha(1a)-AR subtype. TEA was also able to synergistically potentiate the second messenger production by weak partial alpha(1)-AR agonists and this effect was fully inhibited by the alpha(1)-AR antagonist prazosin. However, this synergistic potentiation was not observed for full alpha(1)-AR agonists. Instead, TEA caused a parallel rightward shift of the dose-response curve, consistent with the properties of competitive antagonism. TEA specifically bound to a single population of alpha(1)-ARs with a K-i of 28.7 +/- 4.7 mM. In addition, the site of binding by TEA to the alpha(1)-AR is at the conserved aspartic acid residue in transmembrane domain three, which is part of the constraining salt bridge. These results indicate a direct interaction of TEA in the receptor agonist binding pocket that leads to a disruption of the constraining salt bridge, thereby initiating alpha(1)-AR activation.

Patients on H2-receptor antagonists--are we investigating them?

A survey was made of patients receiving H2-receptor antagonists in a group practice serving 8600 patients. Two hundred and twelve patients (2%) who had received at least one prescription for H2 antagonists in a 12 month period were identified. When compared with the practice population, men and patients over 50 years old were more likely to be taking these drugs (P less than 0.01 and P less than 0.001, respectively). One hundred and fifty-seven patients (74%) were investigated before commencing therapy; 114 (73%) of these patients were investigated via the hospital outpatient department, despite the general practitioners having full open access to barium meals. Only 23 (15%) of the patients investigated were found to have no active pathology. Twenty-nine (14%) of the 212 study patients had received one or more gastrointestinal investigations in the 18 months subsequent to starting H2-antagonist therapy. Twenty-five of these patients had also received an investigation before starting therapy. One hundred and eleven patients (52%) had had their H2 antagonist therapy initiated by their general practitioner.

flp-32 Ligand/Receptor Silencing Phenocopy Faster Plant Pathogenic Nematodes

Restrictions on nematicide usage underscore the need for novel control strategies for plant pathogenic nematodes such as Globodera pallida (potato cyst nematode) that impose a significant economic burden on plant cultivation activities. The nematode neuropeptide signalling system is an attractive resource for novel control targets as it plays a critical role in sensory and motor functions. The FMRFamide-like peptides (FLPs) form the largest and most diverse family of neuropeptides in invertebrates, and are structurally conserved across nematode species, highlighting the utility of the FLPergic system as a broad-spectrum control target. flp-32 is expressed widely across nematode species. This study investigates the role of flp-32 in G. pallida and shows that: (i) Gp-flp-32 encodes the peptide AMRNALVRFamide; (ii) Gp-flp-32 is expressed in the brain and ventral nerve cord of G. pallida; (iii) migration rate increases in Gp-flp-32-silenced worms; (iv) the ability of G. pallida to infect potato plant root systems is enhanced in Gp-flp-32-silenced worms; (v) a novel putative Gp-flp-32 receptor (Gp-flp-32R) is expressed in G. pallida; and, (vi) Gp-flp-32R silenced worms also display an increase in migration rate. This work demonstrates that Gp30 flp-32 plays an intrinsic role in the modulation of locomotory behaviour in G. pallida, and putatively interacts with at least one novel G-protein coupled receptor (Gp-flp-32R). This is the first functional characterisation of a parasitic nematode FLP-GPCR. © 2013 Atkinson et al.

Increased expression of bronchial epithelial transient receptor potential vanilloid 1 channels in severe asthma

BACKGROUND: The airway epithelium is exposed to a range of physical and chemical irritants in the environment that are known to trigger asthma. Transient receptor potential (TRP) cation channels play a central role in sensory responses to noxious physical and chemical stimuli. Recent genetic evidence suggests an involvement of transient receptor potential vanilloid 1 (TRPV1), one member of the vanilloid subfamily of TRP channels, in the pathophysiology of asthma. The functional expression of TRPV1 on airway epithelium has yet to be elucidated.

OBJECTIVE: In this study we examined the molecular, functional, and immunohistochemical expression of TRPV1 in asthmatic and healthy airways.

METHODS: Bronchial biopsy specimens and bronchial brushings were obtained from healthy volunteers (n = 18), patients with mild-to-moderate asthma (n = 24), and patients with refractory asthma (n = 22). Cultured primary bronchial epithelial cells from patients with mild asthma (n = 4), nonasthmatic coughers (n = 4), and healthy subjects (n = 4) were studied to investigate the functional role of TRPV1.

RESULTS: Quantitative immunohistochemistry revealed significantly more TRPV1 expression in asthmatic patients compared with healthy subjects, with the greatest expression in patients with refractory asthma (P = .001). PCR and Western blotting analysis confirmed gene and protein expression of TRPV1 in cultured primary bronchial epithelial cells. Patch-clamp electrophysiology directly confirmed functional TRPV1 expression in all 3 groups. In functional assays the TRPV1 agonist capsaicin induced dose-dependent IL-8 release, which could be blocked by the antagonist capsazepine. Reduction of external pH from 7.4 to 6.4 activated a capsazepine-sensitive outwardly rectifying membrane current.

CONCLUSIONS: Functional TRPV1 channels are present in the human airway epithelium and overexpressed in the airways of patients with refractory asthma. These channels might represent a novel therapeutic target for the treatment of uncontrolled asthma.