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Cystic fibrosis (CF) causing mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) lead to mislocalization of CFTR protein from the brush border membrane of epithelial tissues and/or its dysfunction as a chloride channel. In initial reports, it was proposed that certain channels from the ClC family of chloride channels may provide compensatory or alternative pathways for epithelial chloride secretion in tissues from cystic fibrosis patients. In the present work, we provide the first evidence that ClC-4 protein is functionally expressed on the surface of the intestinal epithelium and hence, is appropriately localized to act as a therapeutic target in this CF-affected tissue. We show using confocal and electron microscopy that ClC-4 co-localizes with CFTR in the brush border membrane of the epithelium lining intestinal crypts in mouse and human tissues. In Caco-2 cells, a cell line thought to model human enterocytes, ClC-4 protein is expressed on the cell surface and also partially co-localizes with EEA1 and transferrin, marker molecules of early and recycling endosomes, respectively. Hence, like CFTR, ClC-4 may cycle between the plasma membrane and endosomal compartment. Furthermore, we show that ClC-4 functions as a chloride channel on the surface of these epithelial cells as antisense ClC-4 cDNA expression reduced the amplitude of endogenous chloride currents by 50%. These studies provide the first evidence that ClC-4 is endogenously expressed and may be functional in the brush border membrane of enterocytes and hence should be considered as a candidate channel to provide an alternative pathway for chloride secretion in the gastrointestinal tract of CF patients.

It has been previously determined that ClC-2, a member of the ClC chloride channel superfamily, is expressed in certain epithelial tissues. These findings fueled speculation that ClC-2 can compensate for impaired chloride transport in epithelial tissues affected by cystic fibrosis and lacking the cystic fibrosis transmembrane conductance regulator. However, direct evidence linking ClC-2 channel expression to epithelial chloride secretion was lacking. In the present studies, we show that ClC-2 transcripts and protein are present endogenously in the Caco-2 cell line, a cell line that models the human small intestine. Using an antisense strategy we show that ClC-2 contributes to native chloride currents in Caco-2 cells measured by patch clamp electrophysiology. Antisense ClC-2-transfected monolayers of Caco-2 cells exhibited less chloride secretion (monitored as iodide efflux) than did mock transfected monolayers, providing the first direct molecular evidence that ClC-2 can contribute to chloride secretion by the human intestinal epithelium. Further, examination of ClC-2 localization by confocal microscopy revealed that ClC-2 contributes to secretion from a unique location in this epithelium, from the apical aspect of the tight junction complex. Hence, these studies provide the necessary rationale for considering ClC-2 as a possible therapeutic target for diseases affecting intestinal chloride secretion such as cystic fibrosis.

The expression of genes for contractile proteins during myogenesis is coordinately regulated. Uncoupling the expression of the slow/cardiac troponin C (sTnC) gene from this process with an antisense phosphorothioate oligodeoxynucleotide (ODN) was used to examine the presence of any post-transcriptional mechanisms for regulating muscle protein synthesis. Approximately 70 and 50% decreases in sTnC polypeptide synthesis and mRNA levels, respectively, were achieved after 4 days antisense treatment. This decrease in sTnC polypeptide synthesis was not reflected in a similar decline in the steady-state level of this polypeptide. Extension of the ODN treatment to 7 days was required to produce a substantial decrease in the steady-state level of sTnC polypeptide. Our investigation suggests that during the 4-day treatment, the affected cells stabilized the sTnC polypeptide level by increasing its half-life. However, the stabilizing effect appears to be overridden during prolonged (7 days) antisense ODN treatment. Measurement of the polypeptide synthesis and mRNA levels of several contractile proteins showed no evidence of cross-regulation among the genes to coordinately regulate their expression levels.

The effect of antisense oligodeoxynucleotide to rat troponin T (TnT) mRNA on its expression in differentiated rat L6 myotubes in culture was examined. The target sequence following the initiation codon was between nucleotides 83 and 97 and is found in all mRNAs produced from the f-TNT gene. Our studies showed that chimeric oligomer with one phosphorothioate linkage at the 3'-end was considerably more resistant to nucleases than was a phosphodiester oligomer. The chimeric oligomer produced50% inhibition of TnT polypeptide synthesis. Synthesis of myosin heavy chain (MHC), troponin I (TnI), and alpha and beta tropomyosins (Tm) was not inhibited by the anti-TnT oligomer. However, synthesis of alpha-actin and troponin C (TnC) was somewhat affected by this treatment. Furthermore, compared with the untreated control myotubes, the steady-state level of TnT mRNA was reduced by approximately 40%-50% in anti-TnT oligomer-treated myotubes. Cellular levels of three other muscle mRNAs, alpha-Tm, s-TnI, and alpha-actin were also reduced by approximately 30%-40%. In contrast, fast TnI, beta-Tm, and TnC mRNA levels were not significantly affected by this treatment. Therefore, inhibition of TnT synthesis in differentiated myotubes uncoupled the coordinated expression of muscle proteins.

The effect of blocking expression of a specific gene with antisense phosphodiester oligodeoxynucleotides on the coordinate regulation of myogenesis was studied. Different regions of both fast and slow troponin C (TnC) mRNAs were targeted for binding of the antisense oligomer. The 5'-cap region of both mRNAs was found to be the most effective target for inhibiting the expression of these genes. Approximately 40%-60% inhibition of expression of a specific isoform of TnC was achieved. However, inhibition of the TnC expression did not appreciably alter the pattern of myogenesis of mouse C2C12 cells. The differentiated murine muscle cells were able to cope with this reduced level of the target gene expression by antisense phosphodiester oligomers. We have also used a phosphorothioate oligomer targeted against a common sequence within the coding region of both fast and slow TnC mRNAs. This oligomer was found to be ineffective in blocking TnC gene expression.

The check-points that maintain stoichiometric synthesis of muscle proteins were examined by misexpression of slow troponin C (sTnC) in mouse C2 myotubes. The sTnC mRNA was overexpressed in myotubes by transfecting these cells with a plasmid construct containing the constitutive CMV promoter-driven sTnC cDNA. An approximately four-fold increase of sTnC mRNA level in the transfected cells was observed. However, the increased mRNA level did not produce a corresponding increase of the sTnC polypeptide level in transfected cells. Only a modest 1.5-fold increase of the sTnC polypeptide level in the transfected cells was observed. The excess sTnC polypeptide in transfected cells was found in the soluble form which was not complexed with other thin filament proteins. The difference between the increase of sTnC mRNA and the polypeptide levels in transfected cells was not due to inefficient translation of the overexpressed sTnC mRNA. Analyses of the stability of the sTnC polypeptide in the thin filament and in the unassembled soluble forms showed that the excess soluble sTnC polypeptide was degraded more rapidly than the sTnC polypeptide of the thin filament. Analyses of the mRNA and polypeptide levels of several thin filament complements showed no effect of overexpression of the sTnC mRNA.