gw-6471 and pirinixic-acid

Aim of the study. Inhaled ammonium persulphate (AP) reduces non adrenergic, non cholinergic (NANC) relaxation in the guinea pig trachea, as a part of its inflammatory effects. Peroxisome Proliferator-Activated Receptor (PPAR) stimulation has shown anti-inflammatory properties. This study aimed at evaluating whether the PPAR-α agonist WY 14643 can prevent the reduction in NANC relaxation caused by inhaled AP in the guinea pig trachea. Materials and Methods. Four groups of ten male guinea pigs were treated for three weeks with inhaled AP (10 mg/m3, 30 min per day, group A), saline (group B), AP and WY 14643 (0.36 μM/die, per os, group C), and AP, WY 14643 and the PPAR-α antagonist GW 6471 (0.36 μM/die, per os, group D). NANC relaxations to electrical field stimulation (EFS) at 3 Hz were evaluated in whole tracheal segments as intraluminal pressure changes. Results. The tracheal NANC relaxations were reduced by 90.3% in group A, as compared to group B. In group C, they were reduced by only 22.2%. In group D, they were reduced by 92.6 %. PPAR-α receptors were detected in inhibitory nerve fibers within the trachea as shown by immonohistochemical analysis. Conclusions. The PPAR-α agonist WY 14643 protects the NANC inhibitory system of the guinea pig trachea from the effect of inhaled ammonium persulphate and its protective effect is antagonized by GW 6471. PPAR-α might be exploited.. Scopo dello studio. L’inalazione di ammonio persolfato (AP) riduce il rilasciamento non adrenergico, non colinergico (NANC) nella trachea di cavia, come parte dei suoi effetti infiammatori. La stimolazione dei Peroxisome Proliferator- Activated Receptor (PPAR) ha mostrato di possedere effetti antiinfiammatori. Questo studio ha avuto lo scopo di valutare se l’agonista per i PPAR-α WY 14643 sia in grado di prevenire la riduzione del rilasciamento NANC causata dall’inalazione di AP nella trachea di cavia. Materiali e metodi. Quattro gruppi di dieci cavie maschio sono stati trattati con l’inalazione di AP (10 mg/m3, 30 minuti al giorno, gruppo A), con soluzione fisiologica (gruppo B), con AP e WY 14643 (0.36 μM/die, per os, gruppo C), e con AP, WY 14643 e con l’antagonista PPAR-α GW 6471 (0.36 μM/die, per os, group D). I rilasciamenti NANC indotti dalla stimolazione con campo elettrico, alla frequenza di 3 Hz, sono stati valutati immunodecome variazioni di pressione intraluminale in segmenti tracheali interi. Risultati. I rilasciamenti NANC tracheali erano ridotti del 90.3% nel gruppo A, a confronto con il gruppo B. Nel gruppo C essi erano ridotti solo del 22.2%. Nel gruppo D i rilasciamenti erano ridotti del 92.6%. È stata rilevata la presenza di recettori PPAR-α nelle fibre nervosa inibitorie intrinseche della trachea, mediante analisi immunoistochimica. Conclusioni. L’agonista per i PPAR-α WY 14643 protegge il sistema NANC inibitorio della tracea di cavia dall’effetto dell’inalazione di ammonio persolfato e il suo effetto protettivo è antagonizzato dall’antagonista specifico GW 6471. I PPAR-α potrebbero essere sfruttati come bersaglio farmacologico nella terapia dell’asma.

Topics: Administration, Inhalation; Adrenergic beta-Agonists; Ammonium Sulfate; Animals; Electric Stimulation; Guinea Pigs; Isoproterenol; Male; Muscle Relaxation; Nerve Fibers; Oxazoles; Pilot Projects; PPAR alpha; Pyrimidines; Random Allocation; Trachea; Tyrosine

Head and neck paragangliomas (HNPGLs) are rare tumors that may cause important morbidity, because of their tendency to infiltrate the skull base. At present, surgery is the only therapeutic option, but radical removal may be difficult or impossible. Thus, effective targets and molecules for HNPGL treatment need to be identified. However, the lack of cellular models for this rare tumor hampers this task. PPARα receptor activation was reported in several tumors and this receptor appears to be a promising therapeutic target in different malignancies. Considering that the role of PPARα in HNPGLs was never studied before, we analyzed the potential of modulating PPARα in a unique model of HNPGL cells. We observed an intense immunoreactivity for PPARα in HNPGL tumors, suggesting that this receptor has an important role in HNPGL. A pronounced nuclear expression of PPARα was also confirmed in HNPGL-derived cells. The specific PPARα agonist WY14643 had no effect on HNPGL cell viability, whereas the specific PPARα antagonist GW6471 reduced HNPGL cell viability and growth by inducing cell cycle arrest and caspase-dependent apoptosis. GW6471 treatment was associated with a marked decrease of CDK4, cyclin D3 and cyclin B1 protein expression, along with an increased expression of p21 in HNPGL cells. Moreover, GW6471 drastically impaired clonogenic activity of HNPGL cells, with a less marked effect on cell migration. Notably, the effects of GW6471 on HNPGL cells were associated with the inhibition of the PI3K/GSK3β/β-catenin signaling pathway. In conclusion, the PPARα antagonist GW6471 reduces HNPGL cell viability, interfering with cell cycle and inducing apoptosis. The mechanisms affecting HNPGL cell viability involve repression of the PI3K/GSK3β/β-catenin pathway. Therefore, PPARα could represent a novel therapeutic target for HNPGL.

Topics: Apoptosis; Blotting, Western; Caspase 3; Caspase 6; Caspase 7; Caspases; Caspases, Initiator; Cell Cycle; Cell Survival; Fluorescent Antibody Technique; Head and Neck Neoplasms; Humans; Immunohistochemistry; Oxazoles; PPAR alpha; Pyrimidines; Tumor Cells, Cultured; Tyrosine; Wound Healing

Chronic tobacco use dramatically increases health burdens and financial costs. Limitations of current smoking cessation therapies indicate the need for improved molecular targets. The main addictive component of tobacco, nicotine, exerts its dependency effects via nicotinic acetylcholine receptors (nAChRs). Activation of the homomeric α7 nAChR reduces nicotine's rewarding properties in conditioned place preference (CPP) test and i.v. self-administration models, but the mechanism underlying these effects is unknown. Recently, the nuclear receptor peroxisome proliferator-activated receptor type-α (PPARα) has been implicated as a downstream signaling target of the α7 nAChR in ventral tegmental area dopamine cells. The present study investigated PPARα as a possible mediator of the effect of α7 nAChR activation in nicotine dependence. Our results demonstrate the PPARα antagonist GW6471 blocks actions of the α7 nAChR agonist PNU282987 on nicotine reward in an unbiased CPP test in male ICR adult mice. These findings suggests that α7 nAChR activation attenuates nicotine CPP in a PPARα-dependent manner. To evaluate PPARα activation in nicotine dependence we used the selective and potent PPARα agonist, WY-14643 and the clinically used PPARα activator, fenofibrate, in nicotine CPP and we observed attenuation of nicotine preference, but fenofibrate was less potent. We also studied PPARα in nicotine dependence by evaluating its activation in nicotine withdrawal. WY-14643 reversed nicotine withdrawal signs whereas fenofibrate had modest efficacy. This suggests that PPARα plays a role in nicotine reward and withdrawal and that further studies are warranted to elucidate its function in mediating the effects of α7 nAChRs in nicotine dependence.

Topics: alpha7 Nicotinic Acetylcholine Receptor; Anesthetics, Local; Animals; Benzamides; Bridged Bicyclo Compounds; Cocaine; Conditioning, Operant; Disease Models, Animal; Fenofibrate; Hypolipidemic Agents; Male; Mice; Mice, Inbred ICR; Nicotine; Nicotinic Agonists; Oxazoles; PPAR alpha; Pyrimidines; Self Administration; Substance Withdrawal Syndrome; Tobacco Use Disorder; Tyrosine

Monoacylglycerol acyltransferase 1 (Mogat1) catalyzes the conversion of monoacylglycerols (MAG) to diacylglycerols (DAG), the precursor of several physiologically important lipids such as phosphatidylcholine, phosphatidylethanolamine and triacylglycerol (TAG). Expression of Mogat1 is tissue restricted and it is highly expressed in the kidney, stomach and adipose tissue but minimally in the normal adult liver. To understand the transcriptional regulation of Mogat1, we characterized the mouse and human Mogat1 promoters in human kidney proximal tubule-2 (HK-2) cells. In-silico analysis revealed several peroxisome proliferator response element (PPRE) binding sites in the promoters of both human and mouse Mogat1. These sites responded to all three peroxisome proliferator activated receptor (PPAR) isoforms such that their respective agonist or antagonist activated or inhibited the expression of Mogat1. PPRE site mutagenesis revealed that sites located at -592 and -2518 are very effective in decreasing luciferase reporter gene activity. Chromatin immunoprecipitation (ChIP) assay using PPARα antibody further confirmed the occupancy of these sites by PPARα. While these assays revealed the core promoter elements necessary for Mogat1 expression, there are additional elements required to regulate its tissue specific expression. Chromosome conformation capture (3C) assay revealed additional cis-elements located ~10-15 kb upstream which interact with the core promoter. These chromosomal regions are responsive to both PPARα agonist and antagonist.

Topics: Acyltransferases; Anilides; Animals; Blotting, Northern; Caco-2 Cells; Cell Line; Cell Line, Tumor; Chromatin Immunoprecipitation; Computational Biology; HT29 Cells; Humans; Kidney Tubules, Proximal; Liver; Mice; Oxazoles; PPAR alpha; Promoter Regions, Genetic; Pyrimidines; Rats; Real-Time Polymerase Chain Reaction; Rosiglitazone; Sulfones; Thiazoles; Thiazolidinediones; Thiophenes; Tyrosine

Dilated cardiomyopathy is the most frequent form of myocardial disease. Many factors contribute to dilated cardiomyopathy, for instance, long-term use of doxorubicin, one of the anthracyclines clinically used for cancer chemotherapy, result in dilated cardiomyopathy and congestive heart failure. However, the mechanism underlining doxorubicin-induced cardiomyocyte is still not fully understood. In this study, we evaluate the effects and their mechanisms of PPARα and PGC-1α pathways in doxorubicin induced mice cardiomyocytes. In vitro, cardiomyocytes isolated from hearts of adult FVB/NJ mice were treated with doxorubicin, GW 6471 (PPARα inhibitors) and WY14643 (PPARα agonists). The expression of PPARα and PGC-1α were detected via western blotting and Quantitative Real-Time PCR methods. Changes in energy and substrate metabolism were analyzed. MTT and flow cytometry were used for cell proliferation and apoptosis analysis. We detected expression of PPARα and PGC-1α was significantly higher in control group than doxorubicin group. Mitochondrial dysfunction was found in doxorubicin group including lower content of high-energy phosphates, significantly decreased mitochondrial ANT transport activity and markedly reduced mitochondrial membrane potential compared with control group. Metabolic remodeling existed in doxorubicin group because of higher concentration of free fatty acid and glucose consumption than of control group. More accumulations of reactive oxygen species were detected in doxorubicin group. The decreased cell viability and increased cell apoptosis observed in doxorubicin group. Severe apoptosis in doxorubicin group was verified by a set of markers including Bax, Bcl-2, cytosolic cytochrome c and caspase-3 up-regulation expression. These findings indicate that the PPARα and PGC-1α are closely involved in energy metabolism remodeling and apoptosis in cardiomyocytes.

Topics: Animals; Antibiotics, Antineoplastic; Apoptosis; Biomarkers; Cell Proliferation; Disease Models, Animal; Doxorubicin; Energy Metabolism; Fatty Acids, Nonesterified; Gene Expression Regulation; Glucose; Humans; Mice; Mitochondria; Myocytes, Cardiac; Oxazoles; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; PPAR alpha; Pyrimidines; Reactive Oxygen Species; Tyrosine; Up-Regulation

We investigated the adipogenic activity of cultured human periosteal-derived cells and studied perioxisome proliferator-activated receptor (PPAR) ligand-mediated differentiation of cultured human periosteal-derived cells into osteoblasts. Periosteal-derived cells expressed adipogenic markers, including CCAAT/enhancer binding protein α (C/EBP- α), C/EBP-δ, aP2, leptin, LPL, and PPARγ. Lipid vesicles were formed in the cytoplasm of periosteal-derived cells. Thus, periosteal-derived cells have potential adipogenic activity. The PPARα and PPARγ agonists, WY14643 and pioglitazone, respectively, did not modulate alkaline phosphatase (ALP) activity in periosteal-derived cells during induced osteoblastic differentiation, however, the PPARα and PPARγ antagonists, GW6471 and T0070907, respectively, both decreased ALP activity in these cells. WY14643 did not affect, whereas pioglitazone enhanced, alizarin red-positive mineralization and calcium content in the periosteal-derived cells. GW6471 and T0070907 both decreased mineralization and calcium content. By RT-PCR, pioglitazone significantly increased ALP expression in periosteal-derived cells between culture day 3 and 2 weeks. Pioglitazone increased Runx2 expression after 3 days, which declined thereafter, but did not alter osteocalcin expression. Both of GW6471 and T0070907 decreased ALP mRNA expression. These results suggest that pioglitazone enhances osteoblastic differentiation of periosteal-derived cells by increasing Runx2 and ALP mRNA expression, and increasing mineralization. GW6471 and T0070907 inhibit osteoblastic differentiation of the periosteal-derived cells by decreasing ALP expression and mineralization in the periosteal-derived cells. In conclusion, although further study will be needed to clarify the mechanisms of PPAR-regulated osteogenesis, our results suggest that PPARγ agonist stimulates osteoblastic differentiation of cultured human periosteal-derived cells and PPARα and PPARγ antagonists inhibit osteoblastic differentiation in these cells.

Topics: Benzamides; Cell Differentiation; Cells, Cultured; Humans; Osteoblasts; Oxazoles; Periosteum; Peroxisome Proliferator-Activated Receptors; Pioglitazone; PPAR alpha; PPAR gamma; Pyridines; Pyrimidines; Reverse Transcriptase Polymerase Chain Reaction; Thiazolidinediones; Tyrosine

Fibrate drugs, the peroxisome proliferator-activated receptor alpha (PPARα) agonists, are widely prescribed for the treatment of hyperlipidemia. The present study examined the effect of fibrate drugs on renal OCT2 activity in a heterologous cell system [Chinese hamster ovary (CHO-K1) cells stably transfected with rabbit (rb) OCT2], LLC-PK1, and intact mouse renal cortical slices. We found that both in the CHO-K1 cells expressing rbOCT2 and in LLC-PK1 cells, fenofibrate significantly inhibited [³H]-MPP⁺ uptake whereas clofibrate and WY14643 had no effect. Surprisingly, the inhibitory effect of fenofibrate was not attenuated by GW6471, a PPARα antagonist, indicating that the inhibitory process observed was via a PPARα-independent pathway. Fenofibrate decreased [³H]-MPP⁺ uptakes through a reduction of the maximal transport (J(max)) but without effect on the transporter affinity (K(t)) corresponding to a decrease in membrane expression of OCT2. Since the inhibitory effect of fenofibrate was not prevented by pretreatment with cycloheximide, its inhibitory action did not involve an inhibition of protein synthesis. Similar to the effect seen in the cell-cultured system, the inhibitory effect of fenofibrate was also observed in intact renal cortical slices. Taken together, our data showed that fenofibrate decreased the activity of OCT2 by reducing the number of functional transporters on the membrane, which is likely to be a PPARα-independent pathway.

Topics: Animals; Biological Transport; Cell Line; Cell Survival; CHO Cells; Cricetinae; Cycloheximide; Down-Regulation; Epithelial Cells; Fenofibrate; Hypolipidemic Agents; Kidney Cortex; Male; Membrane Transport Proteins; Mice; Mice, Inbred ICR; Organic Cation Transport Proteins; Organic Cation Transporter 2; Oxazoles; PPAR alpha; Pyrimidines; Rabbits; Swine; Tyrosine