Publications (11) View all
-
Article: Regulation of the mouse gene encoding TAFI by TNFα: role of NFκB binding site.
[show abstract] [hide abstract]
ABSTRACT: Thrombin-activable fibrinolysis inhibitor (TAFI) is a plasma pro-carboxypeptidase, encoded by the gene CPB2, with roles in both inhibition of fibrinolysis and inflammation. In mice, plasma TAFI levels and hepatic CPB2 mRNA expression were found to increase within 24h after intra-peritoneal lipopolysaccharide (LPS) injection. On the other hand, plasma TAFI in humans decrease in experimental endotoxemia and sepsis and we have previously demonstrated that CPB2 mRNA abundance in human hepatoma cells is decreased by inflammatory cytokines. Here, we have evaluated the effects of TNFα on mouse CPB2 expression. Treatment of primary mouse hepatocytes or the mouse hepatic cell line FL83B with TNFα for 12-48h resulted in increases in CPB2 mRNA abundance of up to 2-fold; mouse TAFI protein levels secreted from FL83B cells increased 2.7-fold after 48h treatment with TNFα. When FL83B cells were transfected with reporter plasmids containing the mouse CPB2 5'-flanking region, treatment with TNFα for 24 and 48h resulted in a 1.5-fold increased mouse CPB2 promoter activity. Mutation of a putative NFκB site not conserved in the human gene ablated the increased promoter activity observed following TNFα treatment. This site binds NFκB as assessed by gel mobility shift assays, and TNFα treatment increases the translocation of NFκB from the cytoplasm to the nucleus of mouse hepatocytes. These results demonstrate that the unique NFκB site in the mouse CPB2 promoter is functional and mediates the upregulation of mouse CPB2 expression by TNFα via increase in NFκB translocation to the nucleus.Cytokine 01/2012; 57(3):389-97. · 3.02 Impact Factor -
Article: Sex-specific extraction of organic anions by the rat liver.
[show abstract] [hide abstract]
ABSTRACT: The capacity for hepatic elimination of some compounds is different in males and females and differential expression of a number of sinusoidal and canalicular transporters exists. However, the specific events underlying the functional differences are not understood. To determine how sex influences sinusoidal and canalicular organic anion transport, bile duct-cannulated livers from mature Sprague-Dawley rats of both sexes were single-pass perfused with saline containing the model organic anions bromosulphophthalein (BSP), carboxyfluorescein (CF), carboxyfluorescein diacetate (CFDA) or 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS). Assay of effluent perfusate anion concentration showed that BSP, but not DIDS, extraction was significantly higher in male versus female rats. At 20 min perfusion with 50 microM BSP the mean effluent concentration was 5.6 and 20.1 microM in, respectively, male and female rats. HPLC confirmed that the effluent perfusate concentration of BSP was higher in female as compared with male rats and was not contributed to by its glutathione conjugate. With 25 microM DIDS, the effluent concentration reached 7.3 (male) and 8.2 microM (female), indicating high extraction efficiency. In contrast to BSP and DIDS, CF extraction was very low (<20%) so that differences between male and females could not be assessed. Biliary BSP and CF excretion were, respectively, 3.5- and 4-fold higher in male rats. Neither sinusoidal efflux nor biliary excretion of CF was sex-dependent with a higher cytoplasmic load of CF (during CFDA perfusion). Our results suggest that differences in sinusoidal uptake are responsible for the sex-specific hepatic excretion of some organic anions.Life Sciences 03/2008; 82(7-8):436-43. · 2.53 Impact Factor -
Article: Interaction of the Ca2+-sensing receptor with the inwardly rectifying potassium channels Kir4.1 and Kir4.2 results in inhibition of channel function.
[show abstract] [hide abstract]
ABSTRACT: The Ca(2+)-sensing receptor (CaR), a G protein-coupled receptor, is expressed in many epithelial tissues including the parathyroid glands, kidney, and GI tract. Although its role in regulating PTH levels and Ca(2+) metabolism are best characterized, it may also regulate salt and water transport in the kidney as demonstrated by recent reports showing association of potent gain-of-function mutations in the CaR with a Bartter-like, salt-wasting phenotype. To determine whether this receptor interacts with novel proteins that control ion transport, we screened a human adult kidney cDNA library with the COOH-terminal 219 amino acid cytoplasmic tail of the CaR as bait using the yeast two-hybrid system. We identified two independent clones coding for approximately 125 aa from the COOH terminus of the inwardly rectifying K(+) channel, Kir4.2. The CaR and Kir4.2 as well as Kir4.1 (another member of Kir4 subfamily) were reciprocally coimmunoprecipitated from HEK-293 cells in which they were expressed, but the receptor did not coimmunoprecipitate with Kir5.1 or Kir1.1. Both Kir4.1 and Kir4.2 were immunoprecipitated from rat kidney extracts with the CaR. In Xenopus laevis oocytes, expression of the CaR with either Kir4.1 or Kir4.2 channels resulted in inactivation of whole cell current as measured by two-electrode voltage clamp, but the nonfunctional CaR mutant CaR(R796W), and that does not coimmunoprecipitate with the channels, had no effect. Kir4.1 and the CaR were colocalized in the basolateral membrane of the distal nephron. The CaR interacts directly with Kir4.1 and Kir4.2 and can decrease their currents, which in turn could reduce recycling of K(+) for the basolateral Na(+)-K(+)-ATPase and thereby contribute to inhibition of Na(+) reabsorption.American journal of physiology. Renal physiology 04/2007; 292(3):F1073-81. · 3.68 Impact Factor -
Article: Modulation of Kir4.2 rectification properties and pHi-sensitive run-down by association with Kir5.1.
[show abstract] [hide abstract]
ABSTRACT: Inwardly rectifying K+ channels (Kir) comprise seven subfamilies that can be subdivided further on the basis of cytosolic pH (pHi) sensitivity, rectification strength and kinetics, and resistance to run-down. Although distinct residues within each channel subunit define these properties, heteromeric association with other Kir subunits can modulate them. We identified such an effect in the wild-type forms of Kir4.2 and Kir5.1 and used this to further understand how the functional properties of Kir channels relate to their structures. Kir4.2 and a Kir4.2-Kir5.1 fusion protein were expressed in HEK293 cells. Inward currents from Kir4.2 were stable over 10 min and pHi-insensitive (pH 6 to 8). Conversely, currents from Kir4.2-Kir5.1 exhibited a pHi-sensitive run-down at slightly acidic pHi. At pHi 7.2, currents in response to voltage steps positive to EK were essentially time independent for Kir4.2 indicating rapid block by Mg2+. Coexpression with Kir5.1 significantly increased the blocking time constant, and increased steady-state outward current characteristic of weak rectifiers. Recovery from blockade at negative potentials was voltage dependent and 2 to 10 times slower in the homomeric channel. These results show that Kir5.1 converts Kir4.2 from a strong to a weak rectifier, rendering it sensitive to pHi, and suggesting that Kir5.1 plays a role in fine-tuning Kir4.2 activity.Biochimica et Biophysica Acta 12/2006; 1758(11):1837-45. · 4.66 Impact Factor -
Article: Loss of Kv and MaxiK currents associated with increased MRP1 expression in small cell lung carcinoma.
[show abstract] [hide abstract]
ABSTRACT: Regulatory volume decrease and exocrine secretion studies suggest a functional relationship between K+ and organic anion efflux. To test the hypothesis that the expression of K+ channels and MRP1 is reciprocally related, we employed the patch clamp and RT-PCR techniques on weakly (H69) and strongly MRP1-expressing (H69AR) small cell lung cancer cells. H69AR cells do not express the time- and voltage-dependent delayed rectifying K+ current (Kv) reported earlier in H69 cells and confirmed here. About 80% of the Kv current in H69 cells inactivated at 0 mV, allowing us to identify other K+ currents present in these cells. Whole-cell currents from cells dialyzed and bathed in K-gluconate as the major ions exhibited inward rectification in both cell types. Inwardly rectifying (Kir) currents in both H69 and H69AR cells showed time-dependent activation and slow inactivation at large negative potentials. H69 cells also express a threefold larger Ca2+ -stimulated K+ -selective and iberiotoxin-sensitive current relative to H69AR cells. In excised inside-out patches exposed to 145 mM symmetrical K+ solutions, H69 cells expressed a voltage- and Ca2+ -sensitive large conductance (128 +/- 5 pS) K+ channel (MaxiK). MaxiK-like currents were not observed at the whole-cell or single-channel level in H69AR cells. RT-PCR identified MaxiKalpha transcripts in H69 but not H69AR cells. These results indicate that two K+ currents (MaxiK and Kv) and the organic anion transporter MRP1 are reciprocally expressed in H69 and H69AR cells.Journal of Cellular Physiology 12/2006; 209(2):535-41. · 3.87 Impact Factor
