Association between Adiponectin and Leptin Receptor Genetic Polymorphisms and Clinical Manifestations of Metabolic Syndrome.

Abdominal obesity coupled with polygenic hereditary defects is considered the initial event in the development of metabolic syndrome (MS). The purpose of this study was to analyse the frequency with which polymorphic loci of adiponectin (ADIPOQ) and leptin (LEP) genes occur in patients with MS and the association between the symptoms of MS and these polymorphisms. DNA was isolated from the whole blood of 207 patients with MS and 100 healthy individuals (control group) using the phenol-chloroform method. Gene polymorphisms were determined using real-time polymerase chain reaction (PCR). The most common variant of the ADIPOQ (rs2241766) gene among MS patients was the GT genotype. The A allele of the LEP (rs7799039) gene was found to be the most frequent in MS patients. The highest systolic blood pressure was found in carriers of the GG genotype of the LEP (rs7799039) gene. The carriers of the ADIPOQ (rs2241766) GT genotype were associated with the highest systolic blood pressure and body mass index (BMI); carriers of the ADIPOQ (rs2241766) GG genotype were associated with the highest diastolic blood pressure, hyperglycaemia, and elevated glycated haemoglobin (HbA1c). The results of this study allowed us to establish the unique gene variants associated with the risk of developing MS in the Crimean population.

BR-bombesin: a novel bombesin-related peptide from the skin secretion of the Chaco tree frog (Boana raniceps) with physiological gastric effects.

Bombesin mediates several biological activities in the gastrointestinal (GI) tract and central nervous system in mammals, including smooth muscle contraction, secretion of GI hormones and regulation of homeostatic mechanisms. Here, we report a novel bombesin-like peptide isolated from Boana raniceps. Its amino acid sequence, GGNQWAIGHFM-NH2, was identified and structurally confirmed by HPLC, MS/MS and 454-pyrosequencing; the peptide was named BR-bombesin. The effect of BR-bombesin on smooth muscle contraction was assessed in ileum and esophagus, and its anti-secretory activity was investigated in the stomach. BR-bombesin exerted significant contractile activity with a concentration-response curve similar to that of commercially available bombesin in ileum strips of Wistar rats. In esophageal strips, BR-bombesin acted as an agonist, as many other bombesin-related peptides act, although with different behavior compared to the muscarinic agonist carbachol. Moreover, BR-bombesin inhibited stomach secretion by approximately 50% compared to the untreated control group. This novel peptide has 80% and 70% similarity with the 10-residue C-terminal domain of human neuromedin B (NMB) and human gastrin releasing peptide (GRP10), respectively. Molecular docking analysis revealed that the GRP receptor had a binding energy equal to - 7.3 kcal.mol-1 and - 8.5 kcal.mol-1 when interacting with bombesin and BR-bombesin, respectively. Taken together, our data open an avenue to investigate BR-bombesin in disorders that involve gastrointestinal tract motility and acid gastric secretion.

Disruption of neuromedin B receptor improves mitochondrial oxidative phosphorylation capacity in gastrocnemius muscle of female mice.

Neuromedin B (NB), a bombesin-like peptide, exerts its specific actions by binding to the neuromedin B receptor (NBR), a G protein-coupled receptor. Female NBR-knockout (NBR-KO) mice exhibit resistance to diet-induced obesity, without hyperphagia, suggesting possible increase in energy expenditure. Skeletal muscle (SM) is crucial for whole body energy homeostasis, however, the presence of NB-NBR signaling and its effects in SM are unknown. Here, we show that male and female wild type express Nmbr and Nmb mRNA in SM, with higher levels in females. Female NBR-KO gastrocnemius showed increased Myh7 mRNA level, which characterizes type I fibers (oxidative profile). Their permeabilized gastrocnemius fibers, studied by high-resolution respirometry, exhibited higher consumption of O2 coupled to ATP synthesis and unaltered uncoupled respiration. NBR-KO gastrocnemius had higher protein levels of ATP-synthase and Nduf9 mRNA, corresponding to mitochondrial complex I subunit. NBR-KO gastrocnemius exhibited slight increase in mitochondria number, increased thickness of Z line at electron microscopy, and unaltered mitochondrial dynamics markers. Therefore, in the females' gastrocnemius, a predominantly glycolytic SM, the NBR absence promotes changes that favor mitochondrial oxidative phosphorylation capacity. In addition, in L6 myocytes, NB treatment (5 µg/mL/16 h) promoted lower O2 consumption coupled to ATP synthesis, suggesting direct action at SM cells. Altogether, the study reinforces the hypothesis that inhibition of NB-NBR signaling enhances the capacity for oxidative phosphorylation of white SM, encouraging future studies to elucidate their contribution on other types of SM and whole body energy expenditure, which may lead to a new target to drug development for obesity treatment.NEW & NOTEWORTHY This study describes neuromedin B (NB) and NB receptor as new regulators of skeletal muscle mitochondrial function. The white skeletal muscle mitochondrial oxidative phosphorylation capacity was increased by NB receptor genetic disruption in female mice. These findings may contribute to the resistance to diet-induced obesity, previously found in these mice, which requires future studies. Thus, investigations are necessary to clarify if blockade of NB receptor may be an approach to develop drugs to combat obesity.

Bombesin Receptor Family Activation and CNS/Neural Tumors: Review of Evidence Supporting Possible Role for Novel Targeted Therapy.

G-protein-coupled receptors (GPCRs) are increasingly being considered as possible therapeutic targets in cancers. Activation of GPCR on tumors can have prominent growth effects, and GPCRs are frequently over-/ectopically expressed on tumors and thus can be used for targeted therapy. CNS/neural tumors are receiving increasing attention using this approach. Gliomas are the most frequent primary malignant brain/CNS tumor with glioblastoma having a 10-year survival <1%; neuroblastomas are the most common extracranial solid tumor in children with long-term survival<40%, and medulloblastomas are less common, but one subgroup has a 5-year survival <60%. Thus, there is an increased need for more effective treatments of these tumors. The Bombesin-receptor family (BnRs) is one of the GPCRs that are most frequently over/ectopically expressed by common tumors and is receiving particular attention as a possible therapeutic target in several tumors, particularly in prostate, breast, and lung cancer. We review in this paper evidence suggesting why a similar approach in some CNS/neural tumors (gliomas, neuroblastomas, medulloblastomas) should also be considered.

Single-cell transcriptomics of suprachiasmatic nuclei reveal a Prokineticin-driven circadian network.

Circadian rhythms in mammals are governed by the hypothalamic suprachiasmatic nucleus (SCN), in which 20,000 clock cells are connected together into a powerful time-keeping network. In the absence of network-level cellular interactions, the SCN fails as a clock. The topology and specific roles of its distinct cell populations (nodes) that direct network functions are, however, not understood. To characterise its component cells and network structure, we conducted single-cell sequencing of SCN organotypic slices and identified eleven distinct neuronal sub-populations across circadian day and night. We defined neuropeptidergic signalling axes between these nodes, and built neuropeptide-specific network topologies. This revealed their temporal plasticity, being up-regulated in circadian day. Through intersectional genetics and real-time imaging, we interrogated the contribution of the Prok2-ProkR2 neuropeptidergic axis to network-wide time-keeping. We showed that Prok2-ProkR2 signalling acts as a key regulator of SCN period and rhythmicity and contributes to defining the network-level properties that underpin robust circadian co-ordination. These results highlight the diverse and distinct contributions of neuropeptide-modulated communication of temporal information across the SCN.

Ultra-high-frequency radio-frequency acoustic molecular imaging with saline nanodroplets in living subjects.

Molecular imaging is a crucial technique in clinical diagnostics but it relies on radioactive tracers or strong magnetic fields that are unsuitable for many patients, particularly infants and pregnant women. Ultra-high-frequency radio-frequency acoustic (UHF-RF-acoustic) imaging using non-ionizing RF pulses allows deep-tissue imaging with sub-millimetre spatial resolution. However, lack of biocompatible and targetable contrast agents has prevented the successful in vivo application of UHF-RF-acoustic imaging. Here we report our development of targetable nanodroplets for UHF-RF-acoustic molecular imaging of cancers. We synthesize all-liquid nanodroplets containing hypertonic saline that are stable for at least 2 weeks and can produce high-intensity UHF-RF-acoustic signals. Compared with concentration-matched iron oxide nanoparticles, our nanodroplets produce at least 1,600 times higher UHF-RF-acoustic signals at the same imaging depth. We demonstrate in vivo imaging using the targeted nanodroplets in a prostate cancer xenograft mouse model expressing gastrin release protein receptor (GRPR), and show that targeting specificity is increased by more than 2-fold compared with untargeted nanodroplets or prostate cancer cells not expressing this receptor.

Preclinical Evaluation of the Copper-64 Labeled GRPR-Antagonist RM26 in Comparison with the Cobalt-55 Labeled Counterpart for PET-Imaging of Prostate Cancer.

Gastrin-releasing peptide receptor (GRPR) is overexpressed in the majority of prostate cancers. This study aimed to investigate the potential of 64Cu (radionuclide for late time-point PET-imaging) for imaging of GRPR expression using NOTA-PEG2-RM26 and NODAGA-PEG2-RM26. Methods: NOTA/NODAGA-PEG2-RM26 were labeled with 64Cu and evaluated in GRPR-expressing PC-3 cells. Biodistribution of [64Cu]Cu-NOTA/NODAGA-PEG2-RM26 was studied in PC-3 xenografted mice and compared to the biodistribution of [57Co]Co-NOTA/NODAGA-PEG2-RM26 at 3 and 24 h p.i. Preclinical PET/CT imaging was performed in tumor-bearing mice. NOTA/NODAGA-PEG2-RM26 were stably labeled with 64Cu with quantitative yields. In vitro, binding of [64Cu]Cu-NOTA/NODAGA-PEG2-RM26 was rapid and GRPR-specific with slow internalization. In vivo, [64Cu]Cu-NOTA/NODAGA-PEG2-RM26 bound specifically to GRPR-expressing tumors with fast clearance from blood and normal organs and displayed generally comparable biodistribution profiles to [57Co]Co-NOTA/NODAGA-PEG2-RM26; tumor uptake exceeded normal tissue uptake 3 h p.i.. Tumor-to-organ ratios did not increase significantly with time. [64Cu]Cu-NOTA-PEG2-RM26 had a significantly higher liver and pancreas uptake compared to other agents. 57Co-labeled radioconjugates showed overall higher tumor-to-non-tumor ratios, compared to the 64Cu-labeled counterparts. [64Cu]Cu-NOTA/NODAGA-PEG2-RM26 was able to visualize GRPR-expression in a murine PC model using PET. However, [55/57Co]Co-NOTA/NODAGA-PEG2-RM26 provided better in vivo stability and overall higher tumor-to-non-tumor ratios compared with the 64Cu-labeled conjugates.

A comparative PET imaging study of 44gSc- and 68Ga-labeled bombesin antagonist BBN2 derivatives in breast and prostate cancer models.

INTRODUCTION: Radiolabeled peptides play a central role in nuclear medicine as radiotheranostics for targeted imaging and therapy of cancer. We have recently proposed the use of metabolically stabilized GRPR antagonist BBN2 for radiolabeling with 18F and 68Ga and subsequent PET imaging of GRPRs in prostate cancer. The present work studied the impact of 44gSc- and 68Ga-labeled DOTA complexes attached to GRPR antagonist BBN2 on the in vitro GRPR binding affinity, and their biodistribution and tumor uptake profiles in MCF7 breast and PC3 prostate cancer models. METHODS: DOTA-Ava-BBN2 was radiolabeled with radiometals 68Ga and 44gSc. Gastrin-releasing peptide receptor (GRPR) affinities of peptides were assessed in PC3 prostate cancer cells. GRPR expression profiles were studied in human breast cancer tissue samples and MCF7 breast cancer cells. PET imaging of 68Ga- and 44gSc-labeled peptides was performed in MCF7 and PC3 xenografts as breast and prostate cancer models. RESULTS: Radiopeptides [68Ga]Ga-DOTA-Ava-BBN2 and [44gSc]Sc-DOTA-Ava BBN2 were prepared in radiochemical yields of 70-80% (decay-corrected), respectively. High binding affinities were found for both peptides (IC50 = 15 nM (natGa) and 5 nM (natSc)). Gene expression microarray analysis revealed high GRPR mRNA expression levels in estrogen receptor (ER)-positive breast cancer, which was further confirmed with Western blot and immunohistochemistry. However, PET imaging showed only low tumor uptake of both radiotracers in MCF7 xenografts ([68Ga]Ga-DOTA-BBN2 (SUV60min 0.27 ± 0.06); [44gSc]Sc-DOTA-BBN2 (SUV60min 0.20 ± 0.03)). In contrast, high tumor uptake and retention were found for both radiopeptides in PC3 tumors ([68Ga]Ga-DOTA-BBN2 (SUV60min 0.46 ± 0.07); [44gSc]Sc-DOTA-BBN2 (SUV60min 0.51 ± 0.11)). CONCLUSIONS: Comparison of 68Ga- and 44gSc-labeled DOTA-Ava-BBN2 peptides revealed slight but noticeable differences of the radiometal with an impact on the in vitro GRPR receptor binding properties in PC3 cells. No differences were found in their in vivo biodistribution profiles in MCF7 and PC3 xenografts. Radiopeptides [68Ga]Ga-DOTA-Ava-BBN2 and [44gSc]Sc-DOTA-Ava-BBN2 displayed comparable tumor uptake and retention profiles with rapid blood and renal clearance profiles in both tumor models. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: The favorable PET imaging performance of [44gSc]Sc-DOTA-Ava-BBN2 in prostate cancer should warrant the development of an [43Sc]Sc-DOTA-Ava-BBN2 analog for clinical translation which comes with a main γ-line of much lower energy and intensity compared to 44gSc.

Pilot study and bioinformatics analysis of differentially expressed genes in adipose tissues of rats with excess dietary intake.

Excessive adipose tissue accumulation is an increasing health problem worldwide. The present study aimed to determine differentially expressed genes (DEGs) that are associated with the excessive accumulation of adipose tissues by PCR arrays in an excess dietary intake animal model. For this purpose, male Sprague Dawley rats were randomly assigned to 2 groups: Control (given an ordinary diet) and experimental (given twice the amount of the ordinary diet). After 2 months of feeding, the abdominal cavities of the rats from each group were opened, then subcutaneous and visceral adipose tissues were removed. The adipose tissues collected were then used for total RNA extraction and then reverse transcribed to cDNA, which was then used as a template to identify the DEGs of 84 transcripts for rat obesity by RT2 Profiler PCR Arrays. The results showed significant downregulation of bombesin­like receptor 3 (BRS3) and uncoupling protein 1 (UCP1) in visceral adipose tissues of experimental rats compared with those of the control rats, and differential gene expression analysis showed an association with fat cell differentiation and regulation of triglyceride sequestration, as well as fatty acid binding. The gene expression patterns observed in the present study, which may be associated with peroxisome proliferator­activated receptor­Î³ (PPARG) on excessive visceral adipose tissue accumulation, may be useful in identifying a group of surrogate biomarkers for the early diet­induced accumulation of visceral adipose tissue detection in humans. The biomarkers can also be the specific targets for drug development to reduce excessive visceral adipose tissue accumulation in the body and its associated diseases.

Neuropeptide bombesin receptor activation stimulates growth of lung cancer cells through HER3 with a MAPK-dependent mechanism.

Despite recent advances in treatment of non-small cell lung cancer (NSCLC), prognosis still remains poor and new therapeutic approaches are needed. Studies demonstrate the importance of the EGFR/HER-receptor family in NSCLC growth, as well as that of other tumors. Recently, HER3 is receiving increased attention because of its role in drug resistance and aggressive growth. Activation of overexpressed G-protein-coupled receptors (GPCR) can also initiate growth by transactivating EGFR/HER-family members. GPCR transactivation of EGFR has been extensively studied, but little is known of its ability to transactivate other EGFR/HER-members, especially HER3. To address this, we studied the ability of bombesin receptor (BnR) activation to transactivate all EGFR/HER-family members and their principal downstream signaling cascades, the PI3K/Akt- and MAPK/ERK-pathways, in human NSCLC cell-lines. In all three cell-lines studied, which possessed EGFR, HER2 and HER3, Bn rapidly transactivated EGFR, HER2 and HER3, as well as Akt and ERK. Immunoprecipitation studies revealed Bn-induced formation of both HER3/EGFR- and HER3/HER2-heterodimers. Specific EGFR/HER3 antibodies or siRNA-knockdown of EGFR and HER3, demonstrated Bn-stimulated activation of EGFR/HER members is initially through HER3, not EGFR. In addition, specific inhibition of HER3, HER2 or MAPK, abolished Bn-stimulated cell-growth, while neither EGFR nor Akt inhibition had an effect. These results show HER3 transactivation mediates all growth effects of BnR activation through MAPK. These results raise the possibility that targeting HER3 alone or with GPCR activation and its signal cascades, may be a novel therapeutic approach in NSCLC. This is especially relevant with the recent development of HER3-blocking antibodies.

Impact of natural neuromedin-B receptor variants on iron metabolism.

Iron overload heritability remains partly unexplained. By performing whole exome sequencing in three patients with a clinical phenotype of hemochromatosis not accounted by known genetic risk factors, we identified in all patients rare variants predicted to alter activity of Neuromedin-B receptor (NMBR). Coding NMBR mutations were enriched in 129 patients with hereditary hemochromatosis or iron overload phenotype, as compared to ethnically matched controls, including 100 local healthy blood donors and 1000Genomes project participants (15.5% vs 5%, P = .0038 at burden test), and were associated with higher transferrin saturation in regular blood donors (P = .04). Consistently, in 191 patients with nonalcoholic fatty liver, the most common low-frequency p.L390 M variant was independently associated with higher ferritin (P = .03). In 58 individuals, who underwent oral iron challenge, carriage of the p.L390 M variant was associated with higher transferrin saturation and lower hepcidin release. Furthermore, the circulating concentration of the natural NMBR ligand, Neuromedin-B, was reduced in response to iron challenge. It was also decreased in individuals carrying the p.L390 M variant and with hemochromatosis in parallel with increased transferrin saturation. In mice, Nmbr was induced by chronic dietary iron overload in the liver, gut, pancreas, spleen, and skeletal muscle, while Nmb was downregulated in gut, pancreas and spleen. Finally, Nmb amplified holo-transferrin dependent induction of hepcidin in primary mouse hepatocytes, which was associated with Jak2 induction and abolished by the NMBR antagonist PD168368. In conclusion, NMBR natural variants were enriched in patients with iron overload, and associated with facilitated iron absorption, possibly related to a defect of iron-induced hepcidin release.

Sensitization of spinal itch transmission neurons in a mouse model of chronic itch requires an astrocytic factor.

BACKGROUND: Chronic itch is a highly debilitating symptom among patients with inflammatory skin diseases. Recent studies have revealed that gastrin-releasing peptide (GRP) and its receptor (gastrin-releasing peptide receptor [GRPR]) in the spinal dorsal horn (SDH) play a central role in itch transmission. OBJECTIVE: We aimed to investigate whether GRP-GRPR signaling is altered in SDH neurons in a mouse model of chronic itch and to determine the potential mechanisms underlying these alterations. METHODS: Patch-clamp recordings from enhanced green fluorescent protein (EGFP)-expressing (GRPR+) SDH neurons were used to examine GRP-GRPR signaling in spinal cord slices obtained from Grpr-EGFP mice. Immunohistochemical, genetic (gene expression and editing through adeno-associated virus vectors), and behavioral approaches were also used for in vivo experiments. RESULTS: We observed potentiation of GRP-evoked excitation in the GRPR+ SDH neurons of mice with contact dermatitis, without concomitant changes in GRPR expression. Interestingly, increases in excitation were attenuated by suppressing the reactive state of SDH astrocytes, which are known to be reactive in patients with chronic itch conditions. Furthermore, CRISPR-Cas9-mediated astrocyte-selective in vivo editing of a gene encoding lipocalin-2 (LCN2), an astrocytic factor implicated in chronic itch, suppressed increases in GRP-induced excitation of GRPR+ neurons, repetitive scratching, and skin damage in mice with contact dermatitis. Moreover, LCN2 potentiated GRP-induced excitation of GRPR+ neurons in normal mice. CONCLUSION: Our findings indicate that, under chronic itch conditions, the GRP-induced excitability of GRPR+ SDH neurons is enhanced through a non-cell-autonomous mechanism involving LCN2 derived from reactive astrocytes.

Gastrin-Releasing Peptide (GRP) Stimulates Osteoclastogenesis in Periodontitis.

Periodontitis is a chronic inflammatory disease with alveolar bone resorption and subsequent tooth loss as its ultimate outcomes. Gastrin-releasing peptide (GRP) is a neuropeptide with growth-stimulatory and tumorigenic properties, and neuropeptides have previously been suggested to play a role in the complex cascade of chemical activity associated with periodontal inflammation. In this study, GRP treatment enhanced the differentiation of bone marrow-derived macrophages (BMMs) into osteoclasts, and gastrin-releasing peptide receptor (GRPR) antagonists suppressed the pro-osteoclastogenic effect of GRP. Grpr-siRNA knockdown resulted in a significantly lower number of osteoclasts formed as compared with the control. Interestingly, gene expression analysis indicated downregulation of Grp and Grpr expressions in BMMs during osteoclastogenesis. Moreover, ligature-induced periodontitis model in mice and gingival samples from patients with periodontitis displayed increased immunostaining of GRP in the oral epithelium. Subsequently, stimulation of mouse primary epithelial cells (ECs) and HaCaT cells, human epidermal keratinocytes, with lipopolysaccharides (LPS) of Porphyromonas gingivalis or live P. gingivalis upregulated Grp and Grpr expressions. Finally, coculture of P. gingivalis-stimulated ECs and BMMs using Transwell system revealed that the differentiation of BMMs was induced when subjected to paracrine activation by LPS- as well as live-P. gingivalis stimulated ECs. Taken together, our results demonstrate that the pro-osteoclastogenic properties of BMMs may be modulated by GRP produced by ECs in the periodontal microenvironment.

Development and Characterization of a Novel, High-Affinity, Specific, Radiolabeled Ligand for BRS-3 Receptors.

Bombesin (Bn) receptor subtype 3(BRS-3) is an orphan G-protein-coupled receptor of the Bn family, which does not bind any natural Bn peptide with high affinity. Receptor knockout studies show that the animals develop diabetes, obesity, altered temperature control, and other central nervous system (CNS)/endocrine/gastrointestinal changes. It is present in CNS, peripheral tissues, and tumors; however, its role in normal physiology/pathophysiology, as well as its receptor localization/pharmacology is largely unknown, in part due to the lack of a convenient, specific, direct radiolabeled ligand. This study was designed to address this problem and to develop and characterize a specific radiolabeled ligand for BRS-3. The peptide antagonist Bantag-1 had >10,000-fold selectivity for human BRS-3 (hBRS-3) over other mammalian Bn receptors (BnRs) [i.e., gastrin-releasing peptide receptor (GRPR) and neuromedin B receptor (NMBR)]. Using iodogen and basic conditions, it was radiolabeled to high specific activity (2200 Ci/mmol) and found to bind with high affinity/specificity to hBRS-3. Binding was saturable, rapid, and reversible. The ligand only interacted with known BRS-3 ligands, and not with other specific GRPR/NMBR ligands or ligands for unrelated receptors. The magnitude of 125I-Bantag-1 binding correlated with BRS-3 mRNA expression and the magnitude of activation of phospholipase C in lung cancer cells, as well as readily identifying BRS-3 in lung cancer cells and normal tissues, allowing the direct assessment of BRS-3 receptor pharmacology/numbers on cells containing BRS-3 with other BnRs, which is usually the case. This circumvents the need for subtraction assays, which are now frequently used to assess BRS-3 indirectly using radiolabeled pan-ligands, which interact with all BnRs.

Gastrin-releasing peptide receptor gene silencing inhibits the development of the epithelial-mesenchymal transition and formation of a calcium oxalate crystal in renal tubular epithelial cells in mice with kidney stones via the PI3K/Akt signaling pathway.

Between 1% and 15% of people are globally affected by kidney stones, and this disease has become more common since the 1970s. Therefore, this study aims to investigate the effects of gastrin-releasing peptide receptor (GRPR) gene silencing via the PI3K/Akt signaling pathway on the development of the epithelial-mesenchymal transition (EMT) and formation of a calcium oxalate crystal in renal tubular epithelial cells (TECs) of kidney stones. A total of 70 clean and healthy C57BL/6J mice were assigned into the normal ( n = 10) and kidney stones groups ( n = 60). The underlying regulatory mechanisms of GRPR were analyzed in concert with the treatment of shGRPR-1, LY294002, and shGRPR-1 + LY294002 in TECs isolated from mice with kidney stones. A series of experiments were conducted for the measurement of urinary oxalate and urinary calcium, the renal calcium salt deposition, the positive rate of GRPR, the expressions of renal TECs related genes and calcium oxalate regulation related genes, and the growth of calcium crystals induced by cells. After treatment of shGRPR-1 and shGRPR-1 + LY294002, levels of urinary oxalate and urinary calcium in the serum, as well as positive rate of GRPR, became relatively low, levels of E-cadherin enhanced, whereas levels of Akt, PI3K, GRPR, extents of PI3K and Akt phosphorylation, α-SMA, Vimentin and FSP-1, OPN, MCP-1, and CD44 decreased and a number of crystals reduced. Taken together, we conclude that GRPR gene silencing suppresses the development of the EMT and formation of the calcium oxalate crystal in renal TECs of kidney stones through the inactivation of the PI3K/Akt signaling pathway.

Bombesin Receptor Subtype-3 in Human Diseases.

This review summarizes the recent findings of the roles of bombesin receptor subtype-3 (BRS-3) in various patho-physiological conditions. Studies have demonstrated that two mammalians bombesin-like peptides, GRP and NMB, exhibit a large range of functions by binding to three receptors. Knockout studies showed that the mice BRS-3 has important effects on tumor growth, energy homeostasis, glucose regulation, satiety, and lung development (1,7). BRS-3 is an orphan receptor whose natural ligand is unknown. However, several agonists and antagonists have been synthesized which facilitate its characterization, (D-Tyr6, ß-Ala11, Phe13, Nle14) Bn-(6-14) and MK-5046 are agonists, whereas ML-18 and Bantag-1 are antagonists. With the development of several selective, high-affinity BRS-3 agonists and antagonists, recent studies provided some insights into the biological effects of BRS-3 in several disease states including lung cancer, obesity, diabetes mellitus, asthma, and kidney diseases.

Design, synthesis and in vitro evaluation of heterobivalent peptidic radioligands targeting both GRP- and VPAC1-Receptors concomitantly overexpressed on various malignancies - Is the concept feasible?

Radiolabeled heterobivalent peptidic ligands (HBPLs), being able to address different receptors, are highly interesting tumor imaging agents as they can offer multiple advantages over monovalent peptide receptor ligands. However, few examples of radiolabeled HBPLs have been described so far. One promising approach is the combination of gastrin-releasing peptide receptor (GRPR)- and vasoactive intestinal peptide receptor subtype 1 (VPAC1R)-targeting peptides into one single radioligand since gastrinomas, prostate and breast cancer have been shown to concomitantly or complementarily overexpress both receptors. Here we report the design and synthesis of different HBPLs, comprising a GRPR-binding (BBN7-14) and a VPAC1R-targeting (PACAP-27) peptide. The heterodimers were varied with regard to the distance between the peptide binders and the steric rigidity of the systems. We radiolabeled the HBPLs 19-23 as well as their monomeric reference standards 26 and 27 with 68Ga, achieving radiochemical yields and purities of 95-99% and non-optimized molar activities of 25-61 GBq/µmol. We tested the stability of the radioligands and further evaluated them in vitro regarding their uptake in different prostate carcinoma cell lines (PC-3, DU-145 and VCaP cells). We found that the heterobivalent substances [68Ga]19 - [68Ga]23 showed comparable uptakes into the tumor cells to those of the respective monomers [68Ga]26 and [68Ga]27, indicating that both peptides are still able to address their target receptors. Furthermore, the obtained results indicate that in case of overall low receptor densities, heterobivalent peptides surpass peptide monomers in tumor cell uptake. Most importantly, it could be shown by blocking studies that both peptide parts of the HBPL [68Ga]19 contributed to tumor cell uptake in VCaP cells, expressing both receptor types. Thus, we describe here the first examples of HBPLs being able to address the GRPR as well as the VPAC1R and have the potential to - by several mechanisms - improve tumor targeting for several malignancies compared to monospecific peptides.

Receptor-specific crosstalk between prostanoid E receptor 3 and bombesin receptor subtype 3.

Bombesin receptor subtype 3 (BRS-3) is a GPCR that is expressed in the CNS, peripheral tissues, and tumors. Our understanding of BRS-3's role in physiology and pathophysiology is limited because its natural ligand is unknown. In an attempt to identify this ligand, we screened toad skin ( Bufo bufo gargarizans Cantor) extracts and identified prostaglandins as putative ligands. In BRS-3-transfected human embryonic kidney (HEK) cells, we found that prostaglandins, with prostaglandin E2 (PGE2) being the most potent, fulfill the pharmacologic criteria of affinity, selectivity, and specificity to be considered as agonists to the BRS-3 receptor. However, PGE2 is unable to activate BRS-3 in different cellular environments. We speculated that EP receptors might be the cause of this cellular selectivity, and we found that EP3 is the receptor primarily responsible for the differential PGE2 effect. Consequently, we reconstituted the HEK environment in Chinese hamster ovary (CHO) cells and found that BRS-3 and EP3 interact to potentiate PGE2 signaling. This potentiating effect is receptor specific, and it occurs only when BRS-3 is paired to EP3. Our study represents an example of functional crosstalk between two distantly related GPCRs and may be of clinical importance for BRS-3-targeted therapies.-Zhang, Y., Liu, Y., Wu, L., Fan, C., Wang, Z., Zhang, X., Alachkar, A., Liang, X., Civelli, O. Receptor-specific crosstalk between prostanoid E receptor 3 and bombesin receptor subtype 3.

A possible new target in lung-cancer cells: The orphan receptor, bombesin receptor subtype-3.

Human bombesin receptors, GRPR and NMBR, are two of the most frequently overexpressed G-protein-coupled-receptors by lung-cancers. Recently, GRPR/NMBR are receiving considerable attention because they act as growth factor receptors often in an autocrine manner in different lung-cancers, affect tumor angiogenesis, their inhibition increases the cytotoxic potency of tyrosine-kinase inhibitors reducing lung-cancer cellular resistance/survival and their overexpression can be used for sensitive tumor localization as well as to target cytotoxic agents to the cancer. The orphan BRS-3-receptor, because of homology is classified as a bombesin receptor but has received little attention, despite the fact that it is also reported in a number of studies in lung-cancer cells and has growth effects in these cells. To address its potential importance, in this study, we examined the frequency/relative quantitative expression of human BRS-3 compared to GRPR/NMBR and the effects of its activation on cell-signaling/growth in 13 different human lung-cancer cell-lines. Our results showed that BRS-3 receptor is expressed in 92% of the cell-lines and that it is functional in these cells, because its activation stimulates phospholipase-C with breakdown of phosphoinositides and changes in cytosolic calcium, stimulates ERK/MAPK and stimulates cell growth by EGFR transactivation in some, but not all, the lung-cancer cell-lines. These results suggest that human BRS-3, similar to GRPR/NMBR, is frequently ectopically-expressed by lung-cancer cells in which, it is functional, affecting cell signaling/growth. These results suggest that similar to GRPR/NMBR, BRS-3 should receive increased attention as possible approach for the development of novel treatments and/or diagnosis in lung-cancer.

Augmented Responses to Ozone in Obese Mice Require IL-17A and Gastrin-Releasing Peptide.

Ozone and obesity both increase IL-17A in the lungs. In mice, obesity augments the airway hyperresponsiveness and neutrophil recruitment induced by acute ozone exposure. Therefore, we examined the role of IL-17A in obesity-related increases in the response to ozone observed in obese mice. Lean wild-type and obese db/db mice were pretreated with IL-17A-blocking or isotype antibodies, exposed to air or ozone (2 ppm for 3 h), and evaluated 24 hours later. Microarray analysis of lung tissue gene expression was used to examine the mechanistic basis for effects of anti-IL-17A. Compared with lean mice, ozone-exposed obese mice had greater concentrations of BAL IL-17A and greater numbers of pulmonary IL-17A+ cells. Ozone-induced increases in BAL IL-23 and CCL20, cytokines important for IL-17A+ cell recruitment and activation, were also greater in obese mice. Anti-IL-17A treatment reduced ozone-induced airway hyperresponsiveness toward levels observed in lean mice. Anti-IL-17A treatment also reduced BAL neutrophils in both lean and obese mice, possibly because of reductions in CXCL1. Microarray analysis identified gastrin-releasing peptide (GRP) receptor (Grpr) among those genes that were both elevated in the lungs of obese mice after ozone exposure and reduced after anti-IL-17A treatment. Furthermore, ozone exposure increased BAL GRP to a greater extent in obese than in lean mice, and GRP-neutralizing antibody treatment reduced obesity-related increases in ozone-induced airway hyperresponsiveness and neutrophil recruitment. Our data indicate that IL-17A contributes to augmented responses to ozone in db/db mice. Furthermore, IL-17A appears to act at least in part by inducing expression of Grpr.