Rosemary Ratcliff

Slowed conduction and ventricular tachycardia after targeted disruption of the cardiac sodium channel gene Scn5a

Voltage-gated sodium channels drive the initial depolarization phase of the cardiac action potential and therefore critically determine conduction of excitation through the heart. In patients, deletions or loss-of-function mutations of the cardiac sodium channel gene, SCN5A, have been associated with a wide range of arrhythmias including bradycardia (heart rate slowing), atrioventricular conduction delay, and ventricular fibrillation. The pathophysiological basis of these clinical conditions is unresolved. Here we show that disruption of the mouse cardiac sodium channel gene, Scn5a, causes intrauterine lethality in homozygotes with severe defects in ventricular morphogenesis whereas heterozygotes show normal survival. Whole-cell patch clamp analyses of isolated ventricular myocytes from adult Scn5a+/− mice demonstrate a ≈50% reduction in sodium conductance. Scn5a+/− hearts have several defects including impaired atrioventricular conduction, delayed intramyocardial conduction, increased ventricular refractoriness, and ventricular tachycardia with characteristics of reentrant excitation. These findings reconcile reduced activity of the cardiac sodium channel leading to slowed conduction with several apparently diverse clinical phenotypes, providing a model for the detailed analysis of the pathophysiology of arrhythmias.

Salmonella typhi uses CFTR to enter intestinal epithelial cells.

Homozygous mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis (CF). In the heterozygous state, increased resistance to infectious diseases may maintain mutant CFTR alleles at high levels in selected populations. Here we investigate whether typhoid fever could be one such disease. The disease is initiated when Salmonella typhi enters gastrointestinal epithelial cells for submucosal translocation. We found that S. typhi, but not the related murine pathogen S. typhimurium, uses CFTR for entry into epithelial cells. Cells expressing wild-type CFTR internalized more S. typhi than isogenic cells expressing the most common CFTR mutation, a phenylalanine deleted at residue 508 (Δ508). Monoclonal antibodies and synthetic peptides containing a sequence corresponding to the first predicted extracellular domain of CFTR inhibited uptake of S. typhi. Heterozygous ΔF508 Cftr mice translocated 86% fewer S. typhi into the gastrointestinal submucosa than wild-type Cftr mice; no translocation occurred in ΔF508 Cftr homozygous mice. The Cftr genotype had no effect on the translocation of S. typhimurium. Immunoelectron microscopy revealed that more CFTR bound to S. typhi in the submucosa of Cftr wild-type mice than in ΔF508 heterozygous mice. We conclude that diminished levels of CFTR in heterozygotes may decrease susceptibility to typhoid fever.

A placebo-controlled study of liposome-mediated gene transfer to the nasal epithelium of patients with cystic fibrosis

Cystic fibrosis (CF) is a common, serious, inherited disease. The major cause of mortality in CF is lung disease, due to the failure of airway epithelial cells to express a functional product of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. A potential treatment for CF lung disease is the expression of CFTR in the airways following gene transfer. We have undertaken a double-blinded, placebo-controlled, clinical study of the transfer of the CFTR cDNA to the nasal epithelium of 12 CF patients. Cationic liposomes complexed with plasmid containing the human CFTR cDNA were administered to eight patients, whilst four patients received placebo. Biopsies of the nasal epithelium taken 7 days after dosing were normal. No significant changes in clinical parameters were observed. Functional expression of CFTR assessed by in vivo nasal potential difference measurements showed transient correction of the CF chloride transport abnormality in two patients (15 days after dosing in one patient). Fluorescence microscopy demonstrated CFTR function ex vivo in cells from nasal brushings. In total, evidence of functional CFTR gene transfer was obtained in six out of the eight treated patients. These results provide proof of concept for liposome-mediated CF gene transfer.

A FUNCTIONAL CFTR PROTEIN IS REQUIRED FOR MOUSE INTESTINAL CAMP-, CGMP- AND CA2+-DEPENDENT HCO3- SECRETION

1 Most segments of the gastrointestinal tract secrete HCO3−, but the molecular nature of the secretory mechanisms has not been identified. We had previously speculated that the regulator for intestinal electrogenic HCO3− secretion is the cystic fibrosis transmembrane regulator (CFTR) channel. To prove this hypothesis, we have now measured HCO3− secretion by pH‐stat titration, and recorded the electrical parameters of in vitro duodenum, jejunum and ileum of mice deficient in the gene for the CFTR protein (‘CF‐mice’) and their normal littermates. 2 Basal HCO3− secretory rates were reduced in all small intestinal segments of CF mice. Forskolin, PGE2, 8‐bromo‐cAMP and VIP (cAMP‐dependent agonists), heat‐stable enterotoxin of Escherichia coli (STa), guanylin and 8‐bromo‐cGMP (cGMP‐dependent agonists) and carbachol (Ca2+ dependent) stimulated both the short‐circuit current (ISC) and the HCO3− secretory rate (JHCO3‐) in all intestinal segments in normal mice, whereas none of these agonists had any effect on JHCO3‐ in the intestine of CF mice. 3 To investigate whether Cl−–HCO3− exchangers, which have been implicated in mediating the response to some of these agonists in the intestine, were similarly active in the small intestine of normal and CF mice, we studied CF gradient‐driven 36Cl− uptake into brush‐border membrane (BBM) vesicles isolated from normal and CF mouse small intestine. Both the time course and the peak value for 4,4’‐diisothiocyanostilbene‐2’,2‐disulphonic acid (DIDS)‐inhibited 36Cl− uptake was similar in normal and CF mice BBM vesicles. 4 In summary, the results demonstrate that the presence of the CFTR channel is necessary for agonist‐induced stimulation of electrogenic HCO3− secretion in all segments of the small intestine, and all three intracellular signal transduction pathways stimulate HCO3− secretion exclusively via activation of the CFTR channel.

Generation and characterization of a ΔF508 cystic fibrosis mouse model

We have generated mice carrying the most common mutation in cystic fibrosis (CF), ΔF508, within the cystic fibrosis (Cftr) gene. Mutant animals show pathological and electrophysiological changes consistent with a CF phenotype. ΔF508−/− mice die from peritonitis and show deficiencies in cAMP–activated electrogenic Cl− transport. These mice produce ΔF508 transcripts and show the temperature–dependent trafficking defect first described for the human ΔF508 CFTR protein. A functional CFTR Cl− channel not demonstrated by null CF mice or present at 37 °C was detected following incubation of epithelial cells at 27 °C. Thus, these mice are an accurate ΔF508 model and will be valuable for testing drugs aimed at overcoming the ΔF508 trafficking defect.

Chloride secretion in response to guanylin in colonic epithelial from normal and transgenic cystic fibrosis mice.

1 Guanylin, a 15 amino acid endogenous gut peptide, increased the short circuit current (SCC) in the epithelium of the mouse colon, but only when applied to the apical and not the basolateral surface. 2 By use of selective blockers of epithelial ion transport and modification of the bathing solution, it was concluded that guanylin increased electrogenic chloride secretion but also had a minor effect on electrogenic sodium absorption. In addition there were small residual currents which remained unresolved. 3 The threshold concentration of guanylin causing a SCC increase was less than 50 nm, but at concentrations 40 times greater no indication of a maximally effective concentration was found. 4 Two guanylin isomers with the same amino acid sequence but with the disulphide bridges joined in an alternate fashion showed no activity. Thus only guanylin with the greatest structural homology to heat stable enterotoxin (STa) showed biological activity. 5 The action of guanylin was virtually eliminated in colonic epithelia from transgenic cystic fibrosis (CF) mice. As these animals lack the chloride channel coded by the CF gene sequence, it is likely that the final effector process in murine colonic epithelia involves the CFTR (cystic fibrosis transmembrane conductance regulator) chloride channel. 6 Opportunistic infections of the gut generating STa lead to diarrhoeal conditions via an action of the toxin on apical guanylin receptors. Thus, as discussed, the CF heterozygote may have a genetic advantage in this circumstance.

Ion-transporting activity in the murine colonic epithelium of normal animals and animals with cystic fibrosis.

Electrogenic ion transport in the isolated co-Ionic epithelium from normal and transgenic mice with cystic fibrosis (CF mice) has been investigated under short-circuit current (Isc) conditions. Normal tissues showed chloride secretion in response to carbachol or forskolin, which was sensitive to the Na-K-2Cl cotransport inhibitor, frusemide. Responses to both agents were maintained for at least 12 h in vitro, but the responses to carbachol changed in format throughout this period. By contrast CF colons failed to show the normal secretory responses to carbachol and forskolin, most preparations showing a decrease in Isc that was immediately reversed by frusemide. In CF colons addition of Ba2+ ions or tetraethylammonium (TEA+) to the apical bathing solution antagonised the reduction in Isc caused by the secretagogues. It is concluded that the reduction in Isc in CF colons is due to electrogenic K+ secretion and this was confirmed by flux studies using rubidium-86. In normal colons exposed to TEA+ the responses to for-skolin were greater, but not significantly so, presumably because the minor K+-secretory responses are dominated by major chloride-secretory responses. Again rubidium-86 fluxes showed an increase of K+ secretion in normal colons receiving forskolin. Since the amiloride-sensitive current was not different in CF and normal colons there was no evidence that the CF mice were stressed in a way that increased mineralocorticoid levels and hence K+ secretion. Knowledge of the phenotype of the colonic epithelium of the CF mouse sets the baseline from which attempts at gene therapy for the gut must be judged.

The genetic advantage hypothesis in cystic fibrosis heterozygotes: a murine study.

1. The delta F508 mutation of the cystic fibrosis (CF) gene is of high frequency in man (1 in 25) and in homozygotes causes cystic fibrosis. It is suggested that cystic fibrosis heterozygotes withstand secretory diarrhoea better than normal individuals and so are genetically advantaged. This hypothesis has been examined by measuring electrogenic chloride secretion in gut epithelia of normal and heterozygous CF mice. 2. Chloride secretory responses of normal and heterozygous colonic epithelia to forskolin, vasoactive intestinal polypeptide (VIP), isoprenaline, cholera toxin, heat‐stable enterotoxin (STa), guanylin, carbachol and lysylbradykinin were examined. No significant differences in responses of tissues of the two genotypes were found. 3. Responses of normal and heterozygous ileal epithelia to forskolin and glucose were investigated. Heterozygous tissues responded as well as normal tissues. 4. Frusemide (furosemide) caused virtually identical inhibition of the chloride secretory responses to forskolin in colonic epithelia of both genotypes. 5. No evidence to support the genetic advantage hypothesis in ileal or colonic epithelia of the null CF mouse has been found, at least for acute responses. If the hypothesis is true then either (a) other non‐cystic fibrosis transmembrane conductance regulator (non‐CFTR) transport processes are involved, (b) prolonged exposure to secretagogues is required, or (c) delta F508 CFTR is responsible for the protective effect.

Calcium-activated chloride conductance is not increased in pancreatic duct cells of CF mice

Calcium-activated anion secretion is elevated in the pancreatic ductal epithelium of transgenic cf/cf mice which lack the cystic fibrosis transmembrane conductance regulator (CFTR). To elucidate whether this effect is due to increased activity of calcium-activated chloride channels, we have studied the relationship between CFTR and calcium-activated chloride currents in pancreatic duct cells isolated from Cambridge cf/cf mice. CFTR chloride currents activated by cAMP were detected in 59% (29/49) of wild-type cells and in 50% (20/40) of heterozygous cells. However, we could not detect any CFTR currents in the homozygous cf/cf cells (0/25). The maximum CFTR current density measured at a membrane potential of 60 mV was 23.5±2.8 pA/pF (n=29) in wild-type cells, and about half that value, i.e. 12.4±1.6 pA/pF (n=20) in heterozygotes (P=0.004). Calcium-activated chloride currents were detected in 73% (24/33) of wild-type, 75% (21/28) of heterozygous and in 58% (7/12) of homozygous cf/cf cells. There was no significant difference between the steady-state calcium-activated current densities in the three genotypic groups; the current measured at 60 mV being 527±162 pA/pF (n=24) from wild-type, 316±35 pA/pF (n=21) from heterozygote and 419±83 pA/pF (n=7) from homozygous cells. Our data suggest that lack of CFTR does not enhance the calcium-activated chloride conductance in murine pancreatic duct cells.

Inactivation of the murine cftr gene abolishes cAMP-mediated but not Ca2+-mediated secretagogue-induced volume decrease in small-intestinal crypts

The cellular volume of crypts isolated from 2- to 3-week-old mouse small intestine has been measured to assess the capacity of the epithelial cells to respond to secretagogues. Vasoactive intestinal polypeptide (VIP) or carbachol, respectively cAMP- and calcium-mediated secretagogues, produced a reduction crypt volume attributed to KCl loss through channels activated by the agonists. Consistent with the participation of separate chloride channels, 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS) blocked the carbacholbut not the VIP-induced volume decrease, whilst gl- ibenclamide abolished the VIP effect without affecting the carbachol-induced volume decrease. Animals homozygous for a disrupted cftr gene, introduced by gene targeting, were also used as the source for crypt isolation. In these CFTR(-/-) crypts, VIP failed to elicit any reduction in cellular volume, while the response to carbachol was indistinguishable from that seen in crypts from age-matched control animals. These results are consistent with murine CFTR being a cAMP-activated chloride channel inhibited by glibenclamide and resistant to DIDS. A separate chloride conductance activated by calcium mobilization in small-intestinal crypts appears to be independent of CFTR.