Pharmacological correction of a defect in PPAR-γ signaling ameliorates disease severity in Cftr-deficient mice

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (encoded by Cftr) that impair its role as an apical chloride channel that supports bicarbonate transport. Individuals with cystic fibrosis show retained, thickened mucus that plugs airways and obstructs luminal organs as well as numerous other abnormalities that include inflammation of affected organs, alterations in lipid metabolism and insulin resistance. Here we show that colonic epithelial cells and whole lung tissue from Cftr-deficient mice show a defect in peroxisome proliferator-activated receptor-gamma (PPAR-gamma, encoded by Pparg) function that contributes to a pathological program of gene expression. Lipidomic analysis of colonic epithelial cells suggests that this defect results in part from reduced amounts of the endogenous PPAR-gamma ligand 15-keto-prostaglandin E(2) (15-keto-PGE(2)). Treatment of Cftr-deficient mice with the synthetic PPAR-gamma ligand rosiglitazone partially normalizes the altered gene expression pattern associated with Cftr deficiency and reduces disease severity. Rosiglitazone has no effect on chloride secretion in the colon, but it increases expression of the genes encoding carbonic anhydrases 4 and 2 (Car4 and Car2), increases bicarbonate secretion and reduces mucus retention. These studies reveal a reversible defect in PPAR-gamma signaling in Cftr-deficient cells that can be pharmacologically corrected to ameliorate the severity of the cystic fibrosis phenotype in mice

Metabolon disruption: a mechanism that regulates bicarbonate transport

Carbonic anhydrases (CA) catalyze the reversible conversion of CO(2) to HCO(3)(−). Some bicarbonate transporters bind CA, forming a complex called a transport metabolon, to maximize the coupled catalytic/transport flux. SLC26A6, a plasma membrane Cl(−)/HCO(3)(−) exchanger with a suggested role in pancreatic HCO(3)(−) secretion, was found to bind the cytoplasmic enzyme CAII. Mutation of the identified CAII binding (CAB) site greatly reduced SLC26A6 activity, demonstrating the importance of the interaction. Regulation of SLC26A6 bicarbonate transport by protein kinase C (PKC) was investigated. Angiotensin II (AngII), which activates PKC, decreased Cl(−)/HCO(3)(−) exchange in cells coexpressing SLC26A6 and AT(1a)-AngII receptor. Activation of PKC reduced SLC26A6/CAII association in immunoprecipitates. Similarly, PKC activation displaced CAII from the plasma membrane, as monitored by immunofluorescence. Finally, mutation of a PKC site adjacent to the SLC26A6 CAB site rendered the transporter unresponsive to PKC. PKC therefore reduces CAII/SLC26A6 interaction, reducing bicarbonate transport rate. Taken together, our data support a mechanism for acute regulation of membrane transport: metabolon disruption

Warming has a larger and more persistent effect than elevated CO2 on growing season soil nitrogen availability in a species-rich grassland

Background and aims The terrestrial biosphere's ability to capture carbon is dependent upon soil nitrogen (N) availability, which might reduce as CO2 increases, but global warming has the potential to offset CO2 effects. Here we examine the interactive impact of elevated CO2 (eCO2) and warming on soil N availability and transformations in a low-fertility native grassland in Tasmania, Australia. Methods Using ion exchange membranes, we examined soil nitrogen availability during the growing season from 2004 to 2010 in the TasFACE experiment. We also estimated soil N transformation rates using laboratory incubations. Results Soil N availability varied strongly over time but was more than doubled by experimental warming of 2°C, an impact that was consistent from the fifth year of the experiment to its conclusion. Elevated CO2 reduced soil N availability by ~28%, although this varied strongly over time. Treatment effects on potential N mineralisation also varied strongly from year to year but tended to be reduced by eCO2 and increased by warming. Conclusions These results suggest that warming should increase soil N availability more strongly than it is suppressed by eCO2 in low fertility grasslands such as this, stimulating terrestrial carbon sinks by preventing eCO2-induced nitrogen limitation of primary productivity

Intrinsic thermodynamics of high affinity inhibitor binding to recombinant human carbonic anhydrase IV

Membrane-associated carbonic anhydrase (CA) isoform IV participates in carbon metabolism and pH homeostasis and is implicated in the development of eye diseases such as retinitis pigmentosa and glaucoma. A series of substituted benzenesulfonamides were designed and their binding affinity to CA IV was determined by fluorescent thermal shift assay and isothermal titration calorimetry (ITC). Compound [(4-chloro-2-phenylsulfanyl-5-sulfamoyl-benzoyl)amino]propyl acetate (19) bound CA IV with the K d of 1.0 nM and exhibited significant selectivity over the remaining 11 human CA isoforms. The compound could be developed as a drug targeting CA IV. Various forms of recombinant CA IV were produced in Escherichia coli and mammalian cell cultures. Comparison of their temperature stability in various buffers and salt solutions demonstrated that CA IV is most stable at slightly alkaline conditions and at elevated sodium sulfate concentrations. High-resolution X-ray crystallographic structures of ortho-Cl and meta-thiazole-substituted benzene sulfonamide in complex with CA IV revealed the position of and interactions between the ligand and the protein. Sulfonamide inhibitor binding to CA IV is linked to several reactions—the deprotonation of the sulfonamide amino group, the protonation of CA–Zn(II)-bound hydroxide at the active site of CA IV, and the compensating reactions of the buffer. The dissection of binding-linked reactions yielded the intrinsic thermodynamic parameters, characterizing the interaction between CA IV and the sulfonamides in the binding-able protonation forms, including Gibbs energy, enthalpy, and entropy, that could be used for the characterization of binding to any CA in the process of drug design