Loss of the normal epicardial to endocardial gradient of cftr mRNA expression in the hypertrophied rabbit left ventricle.

The electrical instability of hypertrophied and failing hearts is caused by delayed repolarisation, which is thought to be due in part to altered levels and/or patterns of expression of ion channel genes. The aim of this study was to investigate changes in the levels and pattern of cystic fibrosis transmembrane conductance regulator (cftr) mRNA expression in a combined pressure and volume overload model of heart failure in the rabbit, using in situ mRNA hybridisation. There was a decrease in cftr mRNA expression, primarily due to a decrease in epicardial expression and, hence, loss of the normal epicardial to endocardial gradient of cftr mRNA expression in the rabbit left ventricle. In contrast there was an increase in atrial natriuretic factor (anf) mRNA expression in the hypertrophied hearts with preferential reexpression in subendocardial regions. The patterns of both cftr and anf mRNA expression in the hypertrophied hearts were similar to those seen in embryonic hearts. This suggests that the reversion to an embryonic pattern of gene expression in cardiac hypertrophy applies to ion channel genes. The loss of the normal transmural gradient of repolarising ion channels is likely to contribute to instability of repolarisation in the hypertrophied heart and hence increased risk of cardiac arrhythmias in patients with heart failure.

Developmental regulation of the gradient of cftr expression in the rabbit heart.

Gradients of ion channels across the left ventricular free wall of the heart have been found for a number of repolarizing ion channels. Amongst these are the cAMP-activated chloride channels encoded by cftr. In this report, we show that the epicardial (higher) to endocardial (lower) gradient of cftr mRNA found in adult rabbit hearts is not present in embryonic hearts. The gradient starts to develop shortly after birth, and over a period of 5-6 weeks increases to the levels found in the adult. This is the first report of the developmental regulation of any cardiac ion channel mRNA gradient.

Molecular and functional distributions of chloride conductances in rabbit ventricle.

The regulation of cardiac electrical activity is critically dependent on the distribution of ion channels in the heart. For most ion channels, however, the patterns of distribution and what regulates these patterns are not well characterized. The most likely candidates for the genes that encode the cAMP- and swelling-activated chloride conductances in the heart are an alternatively spliced variant of CFTR and ClC-3, respectively. In this study we have 1) measured the density of CFTR and ClC-3 mRNA levels across the left ventricular free wall (LVFW) of the rabbit heart using in situ hybridization and 2) measured the corresponding current density of cAMP- and swelling-activated chloride channels in myocytes isolated from subepicardial, midmyocardial, and subendocardial regions of the LVFW. There was a highly significant gradient in the whole cell slope conductance of cAMP-activated chloride currents; normalized slope conductance at 0 mV was 15.7 +/- 1.8 pS/pF (n = 9) in subepicardial myocytes, 7.8 +/- 1.5 pS/pF (n = 11) in midmyocardial myocytes, and 4.9 +/- 1.1 pS/pF (n = 9) in subendocardial myocytes. The level of CFTR mRNA was closely correlated with the density of cAMP-activated chloride conductances in different regions of the heart, with the level of CFTR mRNA being three times higher in the subepicardium than in the subendocardium. The whole cell slope conductance of swelling-activated chloride channel activity, measured 3-5 min after the commencement of cell swelling, was higher in myocytes isolated from the subepicardium than in myocytes isolated from the midmyocardium or subendocardium. In contrast, there was a uniform expression of ClC-3 mRNA across the LVFW of the rabbit heart. These results suggest that the control of gene expression is an important contributor in regulating the distribution of cAMP-activated chloride channels in the rabbit heart but that it may be less important for the swelling-activated chloride channels.

Tissue-specific in vivo transcription start sites of the human and murine cystic fibrosis genes.

The in vivo transcription start sites of the human cystic fibrosis transmembrane conductance regulator gene ( CFTR ) and its murine homologue ( Cftr ) have been mapped in a range of tissues using the technique of 5' rapid amplification of cDNA ends (5' RACE). These are the first in vivo transcription start sites for CFTR or Cftr to be reported. Distinct, tissue-specific patterns of CFTR start site usage were identified in both mouse and human. In particular, striking variation in the position of the murine Cftr transcription start site was seen along the length of the intestinal tract; different start sites being utilized in ileum and in duodenum. In humans, distinct transcription start sites are utilized in adult and foetal lungs. In addition, a novel 5'-untranslated exon of murine Cftr , denoted exon -1, was identified and shown to be expressed exclusively in mouse testis. Expression of exon -1-containing Cftr transcripts was shown by mRNA in situ hybridization to be confined to the germ cells and to be regulated during spermatogenesis.

Complementation of null CF mice with a human CFTR YAC transgene.

We have made transgenic mice carrying a 320 kb YAC with the intact human cystic fibrosis transmembrane regulator (CFTR) gene. Mice that only express the human transgene were obtained by breeding with Cambridge null CF mice. One line has approximately two copies of the intact YAC. Mice carrying this transgene and expressing no mouse cftr appear normal and breed well, in marked contrast to the null mice, where 50% die by approximately 5 days after birth. The chloride secretory responses in these mice are as large or larger than in wild-type tissues. Expression of the transgene is highly cell type specific and matches that of the endogenous mouse gene in the crypt epithelia throughout the gut and in salivary gland tissue. However, there is no transgene expression in some tissues, such as the Brunner's glands, where it would be expected. Where there are differences between the mouse and human pattern of expression, the transgene follows the mouse pattern. We have thus defined a cloned fragment of DNA which directs physiological levels of expression in many of the specific cells where CFTR is normally expressed.

Co-ordinate regulation of the cystic fibrosis and multidrug resistance genes in cystic fibrosis knockout mice.

The cystic fibrosis (Cftr and multidrug resistance (Mdr1) genes encode structurally similar proteins which are members of the ABC transporter superfamily. These genes exhibit complementary patterns of expression in vivo, suggesting that the regulation of their expression may be co-ordinated. We have tested this hypothesis in vivo by examining Cftr and Mdr1 expression in cystic fibrosis knockout transgenic mice (Cftr(tm1CAM)). Cftr mRNA expression in Cftr(tm1CAM)/Cftr(tm1CAM) mice was 4-fold reduced in the intestine, as compared with littermate wild-type mice. All other Cftr(tm1CAM)/Cftr(tm1CAM) mouse tissues examined showed similar reductions in Cftr expression. In contrast, we observed a 4-fold increase in Mdr1 mRNA expression in the intestines of neonatal and 3- to 4-week-old Cftr(tm1CAM)/Cftr(tm1CAM) mice, as compared with age-matched +/+ mice, and an intermediate level of Mdr1 mRNA in heterozygous Cftr(tm1CAM) mice. In 10-week-old, Cftr(tm1CAM)/Cftr(tm1CAM) mice and in contrast to the younger mice, Mdr1 mRNA expression was reduced, by 3-fold. The expression of two control genes, Pgk-1 and Mdr2, was similar in all genotypes, suggesting that the changes in Mdr1 mRNA levels observed in the Cftr(tm1CAM)/Cftr(tm1CAM) mice are specific to the loss of Cftr expression and/or function. These data provide further evidence supporting the hypothesis that the regulation Cftr and Mdr1 expression is co-ordinated in vivo, and that this co-ordinate regulation is influenced by temporal factors.

Properties of the cAMP-activated C1- current in choroid plexus epithelial cells isolated from the rat.

1. This study used whole-cell patch clamp and RNA in situ hybridization experiments to determine whether the cAMP-activated C1- current expressed in choroid plexus epithelial cells was carried by the cystic fibrosis transmembrane conductance regulator (CFTR) channel. 2. In patch clamp experiments, inclusion of 0.25 mM cAMP and 375 protein kinase A catalytic subunit (PKA) in the electrode solution caused activation of an inwardly rectifying current (21/23 cells). This current was C1- selective, since the current reversal potential (Erev) was -31 +/- 3 mV with equilibrium potential values for C1- (EC1) and Na+ (ENa) of -44 and 0 mV, respectively. 3. In anion substitution experiments, the relative anion permeability sequence for the inward rectifier was: I- (3.5) > HCO3-(1.5) = C1-(1.0) > Br-(0.6) > aspartate (0.2). 4. The inward rectifier was sensitive to inhibition by a range of known channel inhibitors, including: glibenclamide (100 microns), DIDS (100 and 500 microns), NPPB (100 microns) and Ba2+ (1 mM). 5. In RNA in situ hybridization experiments, using two independent rat CFTR cRNA probes, expression of CFTR could not be detected in epithelial cells from the rat choroid plexus. 6. In conclusion, the cAMP-dependent whole-cell C1- current present in choroid plexus epithelial cells from the rat has properties which are distinctly different from those of CFTR.

Tamoxifen blocks chloride channels. A possible mechanism for cataract formation.

Tamoxifen is an antiestrogen frequently used in the treatment of breast cancer and is currently being assessed as a prophylactic for those at high risk of developing tumors. We have found that tamoxifen and its derivatives are high-affinity blockers of specific chloride channels. This blockade appears to be independent of the interaction of tamoxifen with the estrogen receptor and therefore reflects an alternative cellular target. One of the clinical side effects of tamoxifen is impaired vision and cataract. Chloride channels in the lens of the eye were shown to be essential for maintaining normal lens hydration and transmittance. These channels were blocked by tamoxifen and, in organ culture, tamoxifen led to lens opacity associated with cataracts at clinically relevant concentrations. These data suggest a molecular mechanism by which tamoxifen can cause cataract formation and have implications for the clinical use of tamoxifen and related antiestrogens.

Developmental expression of mucin genes MUC1 and MUC2.

The mucin gene MUC1, is expressed in a number of human ductal epithelia in vivo including those within the pancreas, mammary gland, kidney and genital ducts. Further it is expressed at a high level in certain tumours and tumour-derived cell lines. MUC2 was initially isolated from a human jejunum cDNA library and is thought to be one of the major intestinal mucin genes, though it is also expressed in the trachea. We have examined the developmental expression of these two mucin genes in human tissues. High level expression of MUC1 has been seen by 12.5 weeks of gestation in the epithelial of the distal respiratory tract and the collecting ducts in the kidney. By 18 weeks MUC1 mRNA is detectable in the colon but pancreatic expression of MUC1 is not seen until late in gestation. MUC2 mRNA is seen by 12 weeks of gestation in the jejunum, ileum and colon, and in large bronchioles of the lung by 18 weeks. The pattern of expression of MUC1 suggests that this mucin may not be involved in early ductal obstruction in the CF pancreas, but both MUC1 and MUC2 may play a role in the development of intestinal disease and MUC1 in early respiratory disease associated with CF.

Expression of the cystic fibrosis gene in human foetal tissues.

In order to examine the onset of the cystic fibrosis (CF) disease process, the expression of the cystic fibrosis gene (CFTR) has been examined in mid-trimester human foetal tissues by in situ hybridization. CFTR mRNA was detected in the epithelia of pancreatic ducts, small intestine, colon, genital ducts, lung and trachea. The majority of these sites of CFTR expression in the foetus are similar to those seen in adult tissues. However, epithelia of the lung, that contain very little CFTR mRNA in the adult, express high levels of CFTR mRNA in the foetus. Since the lung is the major site of pathology and morbidity in CF these findings have implications for treatment.

Correction of the ion transport defect in cystic fibrosis transgenic mice by gene therapy.

Cystic fibrosis (CF) is a lethal inherited disorder affecting about 1 in 2,000 Caucasians. The major cause of morbidity is permanent lung damage resulting from ion transport abnormalities in airway epithelia that lead to mucus accumulation and bacterial colonization. CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that encodes a cyclic-AMP-regulated chloride channel. Cyclic-AMP-regulated chloride conductances are altered in airway epithelia from CF patients, suggesting that the functional expression of CFTR in the airways of CF patients may be a strategy for treatment. Transgenic mice with a disrupted cftr gene are appropriate for testing gene therapy protocols. Here we report the use of liposomes to deliver a CFTR expression plasmid to epithelia of the airway and to alveoli deep in the lung, leading to the correction of the ion conductance defects found in the trachea of transgenic (cf/cf) mice. These studies illustrate the feasibility of gene therapy for the pulmonary aspects of CF in humans.

CFTR expression is regulated during both the cycle of the seminiferous epithelium and the oestrous cycle of rodents.

Severely reduced fertility is a common finding in cystic fibrosis (CF). We used in situ hybridization to examine the cell-specific expression of CFTR in the reproductive organs of rodents. In males CFTR mRNA is found in the round spermatids (spermatogenic stages V-X) and in the principal cells that line the initial segment of the epididymis. In both the testis and the epididymis, CFTR expression is developmentally regulated suggesting that the defect in the genital tract of male CF patients is of developmental origin. CFTR expression in the luminal and glandular epithelium of the uterus is regulated during the oestrous cycle and is maximal at pro-oestrus. Our results provide a biological rationale for the reduced fertility of CF patients, and suggest a possible cause for the comparatively poorer prognosis for women with CF.

The multidrug resistance and cystic fibrosis genes have complementary patterns of epithelial expression.

The cystic fibrosis gene product, CFTR, and the multidrug resistance P-glycoprotein (encoded by the MDR1 gene) are structurally related proteins and both are associated with epithelial chloride channel activities. We have compared their cell-specific expression in the rat by in situ hybridization. In all tissues examined the two genes were found to have complementary patterns of expression, demonstrating exquisite regulation in both cell-specific and temporal fashions. Additionally, a switch in expression from one gene to the other was observed in certain tissues. For example, expression in the intestine switches from CFTR to MDR1 as the cells migrate across the crypt-villus boundary. A switch from CFTR to MDR1 expression was also observed in the uterine epithelium upon pregnancy. These data suggest that CFTR and P-glycoprotein serve analogous roles in epithelial cells and provide additional evidence that P-glycoprotein has a physiological role in regulating epithelial cell volume. The patterns of expression suggest that the regulation of these two genes is coordinately controlled.

Localization of the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) in the rat to chromosome 4 and implications for the evolution of mammalian chromosomes.

We have isolated a partial cDNA encoding the cystic fibrosis transmembrane conductance regulator (CFTR) in the rat. This cDNA hybridizes to a 6.1-kb RNA transcript from the human T84 epithelial cell line and a similarly sized transcript from the rat parotid gland. The nucleotide sequence of this cDNA shows 80.5% identity to the human CFTR cDNA sequence, and the deduced amino acid sequence of rat CFTR shows 75.5% identity to the amino acid sequence of human CFTR. We have used this cDNA to map the location of the gene encoding CFTR to rat chromosome 4. This result places CFTR within a syntenic group on rat chromosome 4 and on human chromosome 7 that includes the genes encoding interleukin 6 (IL6), erythropoietin (EPO), P-glycoprotein 1 (PGY1), and T cell receptor beta chain (TCRB). This group is divided between chromosomes 5 and 6 in the mouse. Mapping of CFTR to rat chromosome 4 shows that this syntenic group has been divided in the mouse lineage during the past 15 million years and further localizes that breakpoint to a sequence homologous to the human chromosome 7q21.1 and 7q32 region. Similarly, a group of five genes, CFTR, TCRB, HOX1, parathyroid hormone-like hormone (PTHLH), and Kirsten rat sarcoma 2 viral (v-Ki-ras2) oncogene homolog (KRAS2), is syntenic on rat chromosome 4 and mouse chromosome 6, but is divided between human chromosomes 7 and 12. These data suggest that the ancestral mammalian chromosome appeared as the present day rat chromosome 4, with all six genes grouped together, and that chromosomal breakages have occurred in the mouse and human lineages since the mammalian divergence.

In vivo cell-specific expression of the cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). The principal manifestations of CF include increased concentration of Cl- in exocrine gland secretions, pancreatic insufficiency, chronic lung disease, intestinal blockage and malabsorption of fat, and male and female infertility. Insight into the function of CFTR can be gained by correlating its cell-specific expression with the physiology of those cells and with CF pathology. Determination of CFTR messenger RNA in rat tissues by in situ hybridization shows that it is specifically expressed in the ductal cells of the pancreas and the salivary glands. In the intestine, decreasing gradients of expression of the CFTR gene are observed on both the crypt-villus and the proximal-distal axes. This expression is consistent with CFTR being responsible for bidirectional Cl- transport, secretion in the intestinal crypts and reabsorption in the silivary gland ducts, and suggests that in these tissues CFTR functions as a regulated Cl- channel. In the lung, a broad band of hybridization includes the mucosa and submucosa of the bronchi and bronchioles. In the testis, CFTR expression is regulated during the cycle of the seminiferous epithelium. Postmeiotic expression is maximal in the round spermatids of stages VII and VIII, suggesting that CFTR plays a critical role in spermatogenesis and that deficiency of this function contributes to CF male infertility.

Evidence for three genes encoding class-I alcohol dehydrogenase subunits in baboon and analysis of the 5' region of the gene encoding the ADH beta subunit.

Five alcohol dehydrogenases (ADH; alcohol: NAD+ oxidoreductase; EC 1.1.1.1) have been identified in the baboon. All are homodimers of five distinct ADH subunits, with the two class-I ADH subunits being differentially expressed in the liver (the beta-subunit) and kidney. We have hybridized restriction-enzyme-digested baboon DNA to a 30-bp probe or a 337-bp DNA fragment, to reveal the presence of three genes encoding class-I ADH subunits in the baboon genome. This result was confirmed by the amplification of three different baboon ADH (bADH) nucleotide (nt) sequences, corresponding to exon 5 in the human gene encoding ADH beta (hADHB) from baboon DNA. Two of these sequences are identical to previously isolated liver and kidney cDNA nt sequences. These results are consistent with a phylogenetic analysis of the nt sequences of class-I hADH and bADH genes. Then, using primers based on the nt sequence of hADHB, we amplified a 336-bp DNA fragment, from genomic DNA, encoding the 5' region of the bADHB gene. In a 49-bp region of overlap, the nt sequence of this DNA fragment was identical to the sequence of a cDNA fragment amplified from baboon liver mRNA, whereas there were seven differences between this DNA fragment and the sequence of a cDNA amplified from baboon kidney mRNA. We used primer extension analysis to identify three adjacent transcriptional start points (tsp) for bADHB mRNA. Initiation of transcription at the most 5' bp leaves a 72-bp untranslated region. Examination of the sequence upstream from the tsp reveals a number of conserved putative regulatory sequence elements.