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In cystic fibrosis (CF), the AF508-CFTR anterograde trafficking from the endoplasmic reticulum to the plasma membrane is inefficient. New strategies for increasing the delivery of [DELTA]F508-CFTR to the apical membranes are thus pathophysiologically relevant targets to study for CF treatment. Recent studies have demonstrated that PDZ-containing proteins play an essential role in determining polarized plasma membrane expression of ionic transporters. In the present study we have hypothesized that the PDZ-containing protein NHE-RF1, which binds to the carboxy terminus of CFTR, rescues AF508-CFTR expression in the apical membrane of epithelial cells. The plasmids encoding [DELTA]F508-CFTR and NHE-RF1 were intranuclearly injected in A549 or Madin-Darby canine kidney (MDCK) cells, and [DELTA]F508-CFFR channel activity was functionally assayed using SPQ fluorescent probe. Cells injected with [DELTA]F508-CFTR alone presented a low chloride channel activity, whereas its coexpression with NHE-RF1 significantly increased both the basal and forskolin-activated chloride conductances. This last effect was lost with [DELTA]F508-CFTR deleted of its 13 last amino acids or by injection of a specific NHE-RF1 antisense oligonucleotide, but not by NHE-RFI sense oligonucleotide. Immunocytochemical analysis performed in MDCK cells transiently transfected with [DELTA]F508-CFTR further revealed that NHE-RF1 specifically determined the apical plasma membrane expression of [DELTA]F508-CFTR but not that of a trafficking defective mutant potassium channel (KCNQ1). These data demonstrate that the modulation of the expression level of CFTR protein partners, like NHE-RF1, can rescue [DELTA]F508-CFTR activity. cystic fibrosis; [DELTA]F508 cystic fibrosis transmembrane conductance regulator; [Na.sup.+]/[H.sup.+] exchanger regulatory factor isoform 1; polarized expression; traffic doi:10.1152/ajplung.00445.2005

Chloride secretion by airway epithelial cells is primordial for water and ion homeostasis and airways surface prevention of infections. This secretion is impaired in several human diseases, including cystic fibrosis, a genetic pathology due to CFTR gene mutations leading to chloride channel defects. A potential therapeutic approach is aiming at increasing chloride secretion either by correcting the mutated CFTR itself or by stimulating non-CFTR chloride channels at the plasma membrane. Here, we studied the role of phospholipase C in regulating the transepithelial chloride secretion in human airway epithelial 16HBE14o- and CFBE cells over-expressing wild type (WT)- or F508del-CFTR. Western blot analysis shows expression of the three endogenous phospholipase C (PLC) isoforms, namely, PLCδ1, PLCγ1, and PLCβ3 in 16HBE14o- cells. In 16HBE14o- cells, we performed Ussing chamber experiments after silencing each of these PLC isoforms or using the PLC inhibitor U73122 or its inactive analogue U73343. Our results show the involvement of PLCβ3 and PLCγ1 in CFTR-dependent short-circuit current activated by forskolin, but not of PLCδ1. In CFBE-WT CFTR and corrected CFBE-F508del CFTR cells, PLCβ3 silencing also inhibits CFTR-dependent current activated by forskolin and UTP-activated calcium-dependent chloride channels (CaCC). Our study supports the importance of PLC in maintaining CFTR-dependent chloride secretion over time, getting maximal CFTR-dependent current and increasing CaCC activation in bronchial epithelial cells. [ABSTRACT FROM AUTHOR]

Bulteau, Laurence, Renaud Derand, Yvette Mettey, Thierry Metaye, M. Rachel Morris, Ceinwen M. McNeilly, Chiara Folli, Luis J. V. Galietta, Olga Zegarra-Moran, Malcolm M. C. Pereira, Chantal Jougla, Robert L. Dormer, Jean-Michel Vierfond, Michel Joffre, and Frederic Becq. Properties of CFTR activated by the xanthine derivative X-33 in human airway Calu-3 cells. Am J Physiol Cell Physiol 279: C1925-C 1937, 2000.--The pharmacological activation of the cystic fibrosis gene protein cystic fibrosis transmembrane conductance regulator (CFTR) was studied in human airway epithelial Calu-3 cells, which express a high level of CFTR protein as assessed by Western blot and in vitro phosphorylation. Immunolocalization shows that CFTR is located in the apical membrane. We performed iodide efflux, whole cell patch-clamp, and short-circuit recordings to demonstrate that the novel synthesized xanthine derivative 3,7-dimethyl-1-isobutylxanthine (X-33) is an activator of the CFTR channel in Calu-3 cells. Whole cell current activated by X-33 or IBMX is linear, inhibited by glibenclamide and diphenylamine-2-carboxylate but not by DIDS or TS-TM calix[4]arene. Intracellular cAMP was not affected by X-33. An outwardly rectifying [Cl.sup.-] current was recorded in the absence of cAMP and X-33 stimulation, inhibited by DIDS and TS-TM calix[4]arene. With the use of short-circuit recordings, X-33 and IBMX were able to stimulate a large concentration-dependent CFTR transport that was blocked by glibenclamide but not by DIDS. Our results show that manipulating the chemical structure of xanthine derivatives offers an opportunity to identify further specific activators of CFTR in airway cells. cystic fibrosis transmembrane conductance regulator; chloride conductance; pharmacology

Activation of G551D CFTR channel with MPB-91: regulation by ATPase activity and phosphorylation. Am J Physiol Cell Physiol 281: C1657-C1666, 2001.--We have designed and synthesized benzo[c]quinolizinium derivatives and evaluated their effects on the activity of G551D cystic fibrosis transmembrane conductance regulator (CFTR) expressed in Chinese hamster ovary and Fisher rat thyroid cells. We demonstrated, using iodide efflux, whole cell patch clamp, and short-circuit recordings, that 5-butyl6-hydroxy-10-chlorobenzo[c]quinolizinium chloride (MPB-91) restored the activity of G551D CFTR (E[C.sub.50] = 85 [micro]M) and activated CFTR in Calu-3 cells (E[C.sub.50] = 47 [micro]M). MPB-91 has no effect on the ATPase activity of wild-type and G551D NBD1/R/GST fusion proteins or on the ATPase, GTPase, and adenylate kinase activities of purified NBD2. The activation of CFTR by MPB-91 is independent of phosphorylation because 1) kinase inhibitors have no effect and 2) the compound still activated CFTR having 10 mutated protein kinase A sites (10SA-CFTR). The new pharmacological agent MPB-91 may be an important candidate drug to ameliorate the ion transport defect associated with CF and to point out a new pathway to modulate CFTR activity. pharmacology; disease-causing mutation; cystic fibrosis; nucleotide binding domains; cystic fibrosis transmembrane conductance regulator

Proteins comprising the first nucleotide-binding- and R-domains of wild-type and ΔF508 cystic fibrosis transmembrane conductance regulator (CFTR) have been synthesised by in vitro transcription/translation. The kinetics and extent of degradation of wild-type and ΔF508 cytoplasmic domain proteins in rabbit reticulocyte lysates, in which proteasome activity was inhibited, were similar, with a half-life of approximately 4 h. The results show for the first time, that the benzo(c)quinolizinium compounds, MPB-07 and MPB-91, selectively inhibit degradation of the ΔF508 cytoplasmic domain protein. Studies using protease inhibitors demonstrated that both ΔF508 and wild-type proteins are substrates for cysteine proteases. The studies provide evidence that benzo(c)quinolizinium compounds protect a proteolytic cleavage site by direct binding to the first cytoplasmic domain of ΔF508-CFTR and this is a likely mechanism for increasing ΔF508-CFTR trafficking in intact cells. [Copyright &y& Elsevier]

Abstract: Cardiac fibroblasts are an integral part of the myocardial tissue and contribute to its remodelling. This study characterises for the first time the calcium-dependent chloride channels (CaCC) in the plasma membrane of primary human atrial cardiac fibroblasts by means of the iodide efflux and the patch clamp methods. The calcium ionophore A23187 and Angiotensin II (Ang II) activate a chloride conductance in cardiac fibroblasts that shares pharmacological similarities with calcium-dependent chloride channels. This chloride conductance is depressed by RNAi-mediated selective Anoctamine 1 (ANO1) but not by Anoctamine 2 (ANO2) which has been revealed as CaCC and is inhibited by the selective ANO1 inhibitor, T16inh-A01. The effect of Ang II on anion efflux is mediated through AT1 receptors (with an EC50 =13.8±1.3nM). The decrease of anion efflux by calphostin C and bisindolylmaleimide I (BIM I) suggests that chloride conductance activation is dependent on PKC. We conclude that ANO1 contributes to CaCC current in human cardiac fibroblasts and that this is regulated by Ang II acting via the AT1 receptor pathway. [Copyright &y& Elsevier]

The syntheses of 4-C-Me-DAB [1,4-dideoxy-1,4-imino-4-C-methyl-d-arabinitol] from l-erythronolactone and of 4-C-Me-LAB [from d-erythronolactone] require only a single acetonide protecting group. The effect of pH on the NMR spectra of 4-C-Me-DAB [pK(a) of the salt around 8.4] is discussed and illustrates the need for care in analysis of both coupling constants and chemical shift. 4-C-Me-DAB (for rat intestinal sucrase K(i) 0.89 μM, IC(50) 0.41 μM) is a competitive - whereas 4-C-Me-LAB (for rat intestinal sucrase K(i) 0.95 μM, IC(50) 0.66 μM) is a non-competitive - specific and potent α-glucosidase inhibitor. A rationale for the α-glucosidase inhibition by DAB, LAB, 4-C-Me-DAB, 4-C-Me-LAB, and isoDAB - but not isoLAB - is provided. Both are inhibitors of endoplasmic reticulum (ER) resident α-glucosidase I and II.

In the disease cystic fibrosis (CF), the most common mutation delF508 results in endoplasmic reticulum retention of misfolded CF gene proteins (CFTR). We show that the alpha-1,2-glucosidase inhibitor miglustat (N-butyldeoxynojirimycin, NB-DNJ) prevents delF508-CFTR/calnexin interaction and restores cAMP-activated chloride current in epithelial CF cells. Moreover, miglustat rescues a mature and functional delF508-CFTR in the intestinal crypts of ileal mucosa from delF508 mice. Since miglustat is an orally active orphan drug (Zavesca) prescribed for the treatment of Gaucher disease, our findings provide the basis for future clinical evaluation of miglustat in CF patients.