Dose-dependent effects of the once-daily GLP-1 receptor agonist lixisenatide in patients with Type 2 diabetes inadequately controlled with metformin:a randomized, double-blind, placebo-controlled trial

To evaluate the dose-response relationship of lixisenatide (AVE0010), a glucagon-like peptide-1 (GLP-1) receptor agonist, in metformin-treated patients with Type 2 diabetes.

Epidermal growth factor receptor (EGFR), HER2 and insulin-like growth factor-1 receptor (IGF-1R) status in ovarian adult granulosa cell tumours

AIMS: Adult granulosa cell tumours (AGCTs) are uncommon ovarian sex cord-stromal tumours which recur following surgical removal in up to 50% of patients. Treatment options for recurrent and advanced stage AGCTs are limited, with poor response to chemotherapy and radiotherapy. We aimed to assess epidermal growth factor receptor (EGFR), HER2 and insulin-like growth factor-1 receptor (IGF-1R) status in AGCTs with a view to investigating whether or not these receptors might be potential therapeutic targets in these neoplasms.

METHODS AND RESULTS: Immunohistochemical staining for EGFR, HER2 and IGF-1R was undertaken in 31 AGCTs. Tumour DNA was also analysed for mutations in the tyrosine kinase domain of EGFR (exons 18-21) by Cobas mutation RT-PCR. Twenty-three of 31 (74%) AGCTs showed some degree of EGFR expression, generally with cytoplasmic or mixed membranous and cytoplasmic staining of variable intensity. Eleven of 27 (41%) cases exhibited strong membranous and cytoplasmic expression of IGF-1R. HER2 expression was not seen. No mutations were found in exons 18-21 of the EGFR gene in hot-spots of therapeutic relevance.

CONCLUSIONS: This study raises the possibility that anti-EGFR and/or anti-IGF-1R therapies may be of potential benefit in ovarian AGCTs, and this requires further study. Lack of known mutations within the tyrosine kinase domain of EGFR suggests that EGFR-related tyrosine kinase inhibitors may not be useful therapeutically.

Neurokinin-1 receptor, a new modulator of lymphangiogenesis in obese-asthma phenotype

Aims Obesity and asthma are widely prevalent and associated disorders. Recent studies of our group revealed that Substance P (SP) is involved in pathophysiology of obese-asthma phenotype in mice through its selective NK1 receptor (NK1-R). Lymphangiogenesis is impaired in asthma and obesity, and SP activates contractile and inflammatory pathways in lymphatics. Our aim was to study whether NK1-R expression was involved in lymphangiogenesis on visceral (VAT) and subcutaneous (SAT) adipose tissues and in the lungs, in obese-allergen sensitized mice. Main methods Diet-induced obese and ovalbumin (OVA)-sensitized Balb/c mice were treated with a selective NK1-R antagonist (CJ 12,255, Pfizer Inc., USA) or placebo. Lymphatic structures (LYVE-1 +) and NK1-R expression were analyzed by immunohistochemistry. A semi-quantitative score methodology was used for NK1-R expression. Key findings Obesity and allergen-sensitization together increased the number of LYVE-1 + lymphatics in VAT and decreased it in SAT and lungs. NK1-R was mainly expressed on adipocyte membranes of VAT, blood vessel areas of SAT, and in lung epithelium. Obesity and allergen-sensitization combined increased the expression of NK1-R in VAT, SAT and lungs. NK1-R antagonist treatment reversed the effects observed in lymphangiogenesis in those tissues. Significance The obese-asthma phenotype in mice is accompanied by increased expression of NK1-R on adipose tissues and lung epithelium, reflecting that SP released during inflammation may act directly on these tissues. Blocking NK1-R affects lymphangiogenesis, implying a role of SP, with opposite physiological consequences in VAT, and in SAT and lungs. Our results provide a clue for a novel SP role in the obese-asthma phenotype.

Neurokinin-1 receptor, a new modulator of lymphangiogenesis in obese-asthma phenotype

Aims: Obesity and asthma are widely prevalent and associated disorders. Recent studies of our group revealed that Substance P (SP) is involved in pathophysiology of obese-asthma phenotype in mice through its selective NK1 receptor (NK1-R). Lymphangiogenesis is impaired in asthma and obesity, and SP activates contractile and inflammatory pathways in lymphatics. Our aim was to study whether NK1-R expression was involved in lymphangiogenesis on visceral (VAT) and subcutaneous (SAT) adipose tissues and in the lungs, in obeseallergen sensitized mice. Main methods: Diet-induced obese and ovalbumin (OVA)-sensitized Balb/c mice were treated with a selective NK1-R antagonist (CJ 12,255, Pfizer Inc., USA) or placebo. Lymphatic structures (LYVE-1+) and NK1-R expression were analyzed by immunohistochemistry. A semi-quantitative score methodology was used for NK1-R expression. Key findings: Obesity and allergen-sensitization together increased the number of LYVE-1+ lymphatics in VAT and decreased it in SAT and lungs. NK1-R was mainly expressed on adipocyte membranes of VAT, blood vessel areas of SAT, and in lung epithelium. Obesity and allergen-sensitization combined increased the expression of NK1-R in VAT, SAT and lungs. NK1-R antagonist treatment reversed the effects observed in lymphangiogenesis in those tissues. Significance: The obese-asthma phenotype in mice is accompanied by increased expression of NK1-R on adipose tissues and lung epithelium, reflecting that SP released during inflammation may act directly on these tissues. Blocking NK1-R affects lymphangiogenesis, implying a role of SP, with opposite physiological consequences in VAT, and in SAT and lungs. Our results provide a clue for a novel SP role in the obese-asthma phenotype.

The neurokinin 1 receptor: a potential new target for anti-platelet therapy?

The important role of platelets in the development of arterial thrombosis and cardiovascular disease is well established. Current treatments for arterial thrombosis include anti-platelet agents such as aspirin, thienopyridines and glycoprotein IIb-IIIa inhibitors. Despite these drugs being effective there remains a substantial unmet clinical demand for more effective therapeutic approaches, which may reflect the existence of alternative underlying regulatory mechanisms to those already targeted. Recent publications have demonstrated a key role for tachykinins in the positive feedback regulation of platelet aggregation and thrombus formation. The pro-thrombotic effects of tachykinins on platelets are mediated through the neurokinin 1 receptor, which may therefore offer a novel therapeutic drug target in the prevention and the treatment of arterial thrombosis.

AT(1) receptor ligands: virtual-screening-based design with TOPP descriptors, synthesis, and biological evaluation of pyrrolidine derivatives

As a continuing effort to establish the structure-activity relationships (SARs) within the series of the angiotensin II antagonists (sartans), a pharmacophoric model was built by using novel TOPP 3D descriptors. Statistical values were satisfactory (PC4: r(2)=0.96, q(2) ((5) (random) (groups))=0.84; SDEP=0.26) and encouraged the synthesis and consequent biological evaluation of a series of new pyrrolidine derivatives. SAR together with a combined 3D quantitative SAR and high-throughput virtual screening showed that the newly synthesized 1-acyl-N-(biphenyl-4-ylmethyl)pyrrolidine-2-carboxamides may represent an interesting starting point for the design of new antihypertensive agents. In particular, biological tests performed on CHO-hAT(1) cells stably expressing the human AT(1) receptor showed that the length of the acyl chain is crucial for the receptor interaction and that the valeric chain is the optimal one.

Domain swapping in the human histamine H-1 receptor

G-protein-coupled receptors (GPCRs) represent the largest family of receptors involved in transmembrane signaling. Although these receptors were generally believed to be monomeric entities, accumulating evidence supports the presence of GPCRs in multimeric forms. Here, using immunoprecipitation as well as time-resolved fluorescence resonance energy transfer to assess protein-protein interactions in living cells, we unambiguously demonstrate the occurrence of dimerization of the human histamine H-1 receptor. We also show the presence of domain-swapped H-1 receptor dimers in which there is the reciprocal exchange of transmembrane domain TM domains 6 and 7 between the receptors present in the dimer. Mutation of aspartate(107) in transmembrane (TM) 3 or phenylalanine(432) in TM6 to alanine results in two radioligand-binding-deficient mutant H-1 receptors. Coexpression of H-1 D(107)A and H-1 F(432)A, however, results in a reconstituted radioligand binding site that exhibits a pharmacological profile that corresponds to the wildtype H-1 receptor. Interestingly, the H-1 receptor radioligands [H-3] mepyramine and [H-3]-(-)- trans-1-phenyl-3-N, N-dimethylamino-1,2,3,4-tetrahydronaphthalene show differential saturation binding values (B-max) for wild-type H-1 receptors but not for the radioligand binding site that is formed upon coexpression of H-1 D(107)A and H-1 F(432)A receptors, suggesting the presence of different H-1 receptor populations.

The role of the melanocortin 3 receptor in mediating the effects of gamma-MSH peptides on the adrenal

The pro-opiomelanocortin (POMC)-derived peptides, pro-gamma-MSH (16K fragment), and Lys-gamma(3)-MSH, have been shown to potentiate the steroidogenic action of corticotrophin (ACTH) on the adrenal cortex. Using a continuously perfused adrenal cell column system, we have tested the hypothesis that gamma-MSH peptides exert their effect through the Melanocortin 3 Receptor (MC3-R), since this is the only known receptor to have high affinity for gamma-MSH peptides and has been suggested to be expressed in the rat adrenal. To investigate this hypothesis we tested whether the MC3-R agonist MTII and antagonist SHU9119 could mimic or block the actions of pro-gamma-MSH. We found that MTII could not mimic, and SHU9119 could not block pro-gamma-MSH mediated potentiation of ACTH-induced steroidogenesis. These results suggest that the MC3-R is not involved in mediating the potentiation effect, adding further evidence to the argument that another melanocortin receptor exists.

Endothelin-converting enzyme-1 regulates trafficking and signalling of the neurokinin 1 receptor in endosomes of myenteric neurones

Neuropeptide signalling at the plasma membrane is terminated by neuropeptide degradation by cell-surface peptidases, and by beta-arrestin-dependent receptor desensitization and endocytosis. However, receptors continue to signal from endosomes by beta-arrestin-dependent processes, and endosomal sorting mediates recycling and resensitization of plasma membrane signalling. The mechanisms that control signalling and trafficking of receptors in endosomes are poorly defined. We report a major role for endothelin-converting enzyme-1 (ECE-1) in controlling substance P (SP) and the neurokinin 1 receptor (NK(1)R) in endosomes of myenteric neurones. ECE-1 mRNA and protein were expressed by myenteric neurones of rat and mouse intestine. SP (10 nM, 10 min) induced interaction of NK(1)R and beta-arrestin at the plasma membrane, and the SP-NK(1)R-beta-arrestin signalosome complex trafficked by a dynamin-mediated mechanism to ECE-1-containing early endosomes, where ECE-1 can degrade SP. After 120 min, NK(1)R recycled from endosomes to the plasma membrane. ECE-1 inhibitors (SM-19712, PD-069185) and the vacuolar H(+)ATPase inhibitor bafilomycin A(1), which prevent endosomal SP degradation, suppressed NK(1)R recycling by >50%. Preincubation of neurones with SP (10 nM, 5 min) desensitized Ca(2+) transients to a second SP challenge after 10 min, and SP signals resensitized after 60 min. SM-19712 inhibited NK(1)R resensitization by >90%. ECE-1 inhibitors also caused sustained SP-induced activation of extracellular signal-regulated kinases, consistent with stabilization of the SP-NK(1)R-beta-arrestin signalosome. By degrading SP and destabilizing endosomal signalosomes, ECE-1 has a dual role in controlling endocytic signalling and trafficking of the NK(1)R: promoting resensitization of G protein-mediated plasma membrane signalling, and terminating beta-arrestin-mediated endosomal signalling.

Protein phosphatase 2A mediates resensitization of the neurokinin 1 receptor

Activated G protein-coupled receptors (GPCRs) are phosphorylated and interact with beta-arrestins, which mediate desensitization and endocytosis. Endothelin-converting enzyme-1 (ECE-1) degrades neuropeptides in endosomes and can promote recycling. Although endocytosis, dephosphorylation, and recycling are accepted mechanisms of receptor resensitization, a large proportion of desensitized receptors can remain at the cell surface. We investigated whether reactivation of noninternalized, desensitized (phosphorylated) receptors mediates resensitization of the substance P (SP) neurokinin 1 receptor (NK(1)R). Herein, we report a novel mechanism of resensitization by which protein phosphatase 2A (PP2A) is recruited to dephosphorylate noninternalized NK(1)R. A desensitizing concentration of SP reduced cell-surface SP binding sites by only 25%, and SP-induced Ca(2+) signals were fully resensitized before cell-surface binding sites started to recover, suggesting resensitization of cell-surface-retained NK(1)R. SP induced association of beta-arrestin1 and PP2A with noninternalized NK(1)R. beta-Arrestin1 small interfering RNA knockdown prevented SP-induced association of cell-surface NK(1)R with PP2A, indicating that beta-arrestin1 mediates this interaction. ECE-1 inhibition, by trapping beta-arrestin1 in endosomes, also impeded SP-induced association of cell-surface NK(1)R with PP2A. Resensitization of NK(1)R signaling required both PP2A and ECE-1 activity. Thus, after stimulation with SP, PP2A interacts with noninternalized NK(1)R and mediates resensitization. PP2A interaction with NK(1)R requires beta-arrestin1. ECE-1 promotes this process by releasing beta-arrestin1 from NK(1)R in endosomes. These findings represent a novel mechanism of PP2A- and ECE-1-dependent resensitization of GPCRs.

Endothelin-converting enzyme 1 promotes re-sensitization of neurokinin 1 receptor-dependent neurogenic inflammation

BACKGROUND AND PURPOSE: The metalloendopeptidase endothelin-converting enzyme 1 (ECE-1) is prominently expressed in the endothelium where it converts big endothelin to endothelin-1, a vasoconstrictor peptide. Although ECE-1 is found in endosomes in endothelial cells, the role of endosomal ECE-1 is unclear. ECE-1 degrades the pro-inflammatory neuropeptide substance P (SP) in endosomes to promote recycling and re-sensitization of its neurokinin 1 (NK(1)) receptor. We investigated whether ECE-1 regulates NK(1) receptor re-sensitization and the pro-inflammatory effects of SP in the endothelium. EXPERIMENTAL APPROACH: We examined ECE-1 expression, SP trafficking and NK(1) receptor re-sensitization in human microvascular endothelial cells (HMEC-1), and investigated re-sensitization of SP-induced plasma extravasation in rats. KEY RESULTS: HMEC-1 expressed all four ECE-1 isoforms (a-d), and fluorescent SP trafficked to early endosomes containing ECE-1b/d. The ECE-1 inhibitor SM-19712 prevented re-sensitization of SP-induced Ca2+ signals in HMEC-1 cells. Immunoreactive ECE-1 and NK(1) receptors co-localized in microvascular endothelial cells in the rat. SP-induced extravasation of Evans blue in the urinary bladder, skin and ears of the rat desensitized when the interval between two SP injections was 10 min, and re-sensitized after 480 min. SM-19712 inhibited this re-sensitization. CONCLUSIONS AND IMPLICATIONS: By degrading endocytosed SP, ECE-1 promotes the recycling and re-sensitization of NK(1) receptors in endothelial cells, and thereby induces re-sensitization of the pro-inflammatory effects of SP. Thus, ECE-1 inhibitors may ameliorate the pro-inflammatory actions of SP.

Ubiquitin-dependent down-regulation of the neurokinin-1 receptor

Transient stimulation with substance P (SP) induces endocytosis and recycling of the neurokinin-1 receptor (NK(1)R). The effects of sustained stimulation by high concentrations of SP on NK(1)R trafficking and Ca(2+) signaling, as may occur during chronic inflammation and pain, are unknown. Chronic exposure to SP (100 nm, 3 h) completely desensitized Ca(2+) signaling by wild-type NK(1)R (NK(1)Rwt). Resensitization occurred after 16 h, and cycloheximide prevented resensitization, implicating new receptor synthesis. Lysine ubiquitination of G-protein-coupled receptors is a signal for their trafficking and degradation. Lysine-deficient mutant receptors (NK(1)RDelta5K/R, C-terminal tail lysines; and NK(1)RDelta10K/R, all intracellular lysines) were expressed at the plasma membrane and were functional because they responded to SP by endocytosis and by mobilization of Ca(2+) ions. SP desensitized NK(1)Rwt, NK(1)RDelta5K/R, and NK(1)RDelta10K/R. However, NK(1)RDelta5K/R and NK(1)RDelta10K/R resensitized 4-8-fold faster than NK(1)Rwt by cycloheximide-independent mechanisms. NK(1)RDelta325 (a naturally occurring truncated variant) showed incomplete desensitization, followed by a marked sensitization of signaling. Upon labeling receptors in living cells using antibodies to extracellular epitopes, we observed that SP induced endocytosis of NK(1)Rwt, NK(1)RDelta5K/R, and NK(1)RDelta10K/R. After 4 h in SP-free medium, NK(1)RDelta5K/R and NK(1)RDelta10K/R recycled to the plasma membrane, whereas NK(1)Rwt remained internalized. SP induced ubiquitination of NK(1)Rwt and NK(1)RDelta5K/R as determined by immunoprecipitation under nondenaturing and denaturing conditions and detected with antibodies for mono- and polyubiquitin. NK(1)RDelta10K/R was not ubiquitinated. Whereas SP induced degradation of NK(1)Rwt, NK(1)RDelta5K/R and NK(1)RDelta10K/R showed approximately 50% diminished degradation. Thus, chronic stimulation with SP induces ubiquitination of the NK(1)R, which mediates its degradation and down-regulation.