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Biomedical subjects

A W Cuthbert

Publications and source records attributed to A W Cuthbert.

At least 37 records · Page 2Linked to original sources
PubMed1998

Electrogenic bicarbonate secretion in mouse gallbladder.

Mouse gallbladders (4 mm2) were investigated using the short-circuit current (Isc) technique. Responses of 50 microA/cm2 were obtained in response to forskolin and agents that stimulated the adenylate cyclase system (IBMX and dibutyryl-cAMP). The calcium ionophore ionomycin increased Isc to 30% of the forskolin-stimulated increase. The forskolin-dependent current was inhibited 40% by acetazolamide but was insensitive to furosemide. Forskolin responses were dependent on the presence of bicarbonate ions; removal from both sides of the membrane or the basolateral side alone caused a significant reduction in responses. Removal of chloride ions from the basolateral side had no effect, while removal from the apical side caused a significant reduction in the forskolin responses, but only by 30%. It is argued that the remaining current (70%) cannot result from a parallel arrangement of a chloride channel and a chloride-bicarbonate exchanger and that bicarbonate is secreted through the apical membrane by a predominantly conductive mechanism. Apparently, forskolin converts a near electrically silent epithelium to an electrogenically secreting tissue.

1-Methyl-3-isobutylxanthine↗
PubMed1998

Restoration by intratracheal gene transfer of bicarbonate secretion in cystic fibrosis mouse gallbladder.

Gallbladders from cystic fibrosis (CF) mice (Cftrtm1Cam and Cftrtm2Cam) were examined with the short-circuit current technique. The tissues failed to show any electrogenic anion transport in response to forskolin (cAMP stimulus) but responded to the Ca2+ ionophore ionomycin. Administration of the plasmid pTrial10-CFTR2 complexed with cationic liposomes (3beta-[N-(dimethylaminoethane)-carbamoyl]cholesterol and L-alpha-phosphatidylethanolamine dioleolyl) to the airways restored the phenotype of CF gallbladders to that of the wild type, but did not do so when given orally. Formation of human CFTR mRNA in gallbladders of transfected CF null mice was demonstrated. Using the reporter genes pCMV-luc and pCMV-LacZ, we showed that 1) the intratracheal route was more effective than the oral,intravenous, intramuscular, subcutaneous, or intraperitoneal routes in expressing luciferase activity in the gallbladder and 2) beta-galactosidase staining after pCMV-LacZ was confined to the columnar epithelium lining the gallbladder without any discernible activity in it smooth muscle. The discovery of an unusual route for gene transfer to the biliary system may give useful insight into counteracting the consequences of biliary fibrosis in human CF patients.

Animals↗
PubMed1997

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.

Animals↗
PubMed1997

Chloride secretion in the trachea of null cystic fibrosis mice: the effects of transfection with pTrial10-CFTR2.

1. An improved novel plasmid backbone, pTrial10, has been developed. We have used this vector to deliver the cDNA for the cystic fibrosis transmembrane conductance regulator (CFTR) to cells, both in vitro and in vivo, complexed with cationic liposomes. 2. Human 293 kidney epithelial cells (HEK 293) showed expression of an immunoprecipitable 165 kDa protein corresponding to CFTR when transfected in vitro with pTrial10-CFTR2, but not when the vector pTrial10 was used. 3. HEK 293 cells transfected with pTrial10-CFTR2, but not pTrial10, demonstrated a cAMP-dependent anion conductance, measured by fluorescence microscopy using a halide-sensitive probe, SPQ. 4. The CFTR-dependent, cAMP-sensitive chloride secretory response in murine tracheal epithelium could be measured if the calcium-dependent chloride secretory process was first maximally stimulated with a mixture of the Ca(2+)-ATPase inhibitor, TBHQ, and the calcium ionophore, A23187. With these conditions wild-type and CF-null (transgenic animals in which the cystic fibrosis (CF) gene has been disrupted so that no CFTR is produced) murine tracheas could be distinguished. The difference between the current elicited by forskolin in wild-type and CF tracheas was highly significantly different (P < 0.001), giving a CFTR-dependent current of 11.2 microA cm-2. 5. Transfection of the airways with pTrial10-CFTR2, but not pTrial10, significantly (P < 0.01) increased the CFTR-dependent chloride secretory current in CF tracheas. The degree of correction was greater when intra-tracheal installation rather than nasal insufflation was used to deliver the plasmids.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1997

Expression of bradykinin B1 receptors in colonic epithelia.

The rat colonic epithelium shows no response to [des-Arg9] bradykinin (DAB) in newly isolated tissues, but does so after 3 h in vitro. The EC50 for DAB is circa 50 nM and the maximal response is 57.8 +/- 5.3 microA cm-2 (n = 68). The whole of the current increase is sensitive to frusemide and is predominantly due to electrogenic chloride secretion. The development of responsiveness is prevented by inhibitors of translation (cycloheximide) and transcription (actinomycin D). Thus the receptor for DAB has the hall marks of a classical B1 kinin receptor. While the classical B2 kinin receptor antagonist HOE140, has no effect on the responses, the classical B1 kinin receptor antagonist, [Leu8]DAB, behaves as an agonist in this system. It is concluded that there may be more than one type of B1 kinin receptor.

Animals↗
PubMed1997

Induction of bradykinin B1 receptors in rat colonic epithelium.

1. Des-Arg9 bradykinin (DAB), a classical B1-kinin receptor agonist was without effect when applied to the basolateral surface of rat isolated colon epithelium. Three hours after tissues were isolated DAB caused, after a delay of up to 2 min, a maintained increase of short circuit current (SCC). 2. The SCC increase in colonic epithelia, mounted in vitro for three hours, caused by DAB was due to electrogenic chloride secretion as the current increase was reversed by frusemide and did not occur in the absence of cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels. The EC50 for DAB was approximately 50 nM. 3. An inhibitor of transcription (actinomycin D) and of translation (cycloheximide) prevented the appearance of DAB sensitivity without affecting the responses to another secretagogue (forskolin). 4. The classical B1-kinin receptor antagonist, Leu8-des-Arg9 bradykinin, was shown to be an agonist in rat colon epithelium. Other B1-kinin receptor antagonists (des-Arg10-Hoe 140 and R-715) inhibited the responses to DAB in 'aged' colonic epithelia, and the inhibition was easily surmounted by increasing the concentration of DAB. 5. Response to DAB did not appear to involve to any significant extent, the formation of prostaglandins, leukotrienes, histamine or nitric oxide. Furthermore, no neuronal involvement was apparent in the stripped colonic preparations. The responses to DAB were not significantly different in epithelia taken from different parts of the distal colon. 6. The differences between the responses of the colonic epithelium to B1- and B2-kinin receptor agonists are discussed.

Animals↗
PubMed1997

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.

Adolescent↗
PubMed1997

A second dose of a CFTR cDNA-liposome complex is as effective as the first dose in restoring cAMP-dependent chloride secretion to null CF mice trachea.

Phase I clinical trials have provided encouraging data suggesting that gene transfer could provide a treatment for cystic fibrosis (CF). However, for all the current viral and nonviral vectors used to deliver the cystic fibrosis transmembrane conductance regulator (CFTR) gene, the duration of CFTR expression is limited, necessitating a repeat dosing regimen to provide a long-term treatment. This study was performed to determine whether a second delivery of a CFTR cDNA-liposome complex could result in a similar level of functional CFTR expression observed after a single delivery and to assess whether the deliveries produced adverse inflammatory responses. CFTR functional expression was assessed by short circuit current measurements of tracheas taken from CF null mice (Cftrtm1Cam) treated with a CFTR cDNA-liposome complex in the upper airways. Mice receiving two deliveries of this complex, the second after the response to the first had declined, showed cAMP-stimulated chloride currents which were not significantly different from normal tracheas or tissues assayed after a single dose of the complex. This double treatment was well tolerated with no discernible inflammation of lung tissue.

Animals↗
PubMed1997

Normalization of ion transport in murine cystic fibrosis nasal epithelium using gene transfer.

The murine nasal epithelium was investigated by the short-circuit current (SCC) technique. Electrogenic sodium absorption was revealed by addition of amiloride and calcium-dependent chloride secretion by the addition of amiloride and calcium-dependent chloride secretion by the addition of 2,5-di-(tert-butyl)-1,4-benzohydroquinone (TBHQ)/ionomycin. In the presence of these agents a further increase in SCC was obtained by addition of forskolin. Epithelia from both cystic fibrosis (CF) null (Cftrtm1Cam) and CF delta F508 (Cftrtm2Cam) mice had enhanced sodium absorption compared with controls, whereas only delta F508 epithelia had increased calcium-dependent chloride secretion. Both strains gave nasal epithelia that showed significantly reduced responses to forskolin, due to the absence of CF transmembrane conductance regulator (CFTR) chloride channels. In Cftrtm2Cam nasal epithelia the forskolin responses were not significantly different from zero. Transfection of these mice with the plasmid pTRIAL10-CFTR2 complexed with cationic liposomes normalized the transporting activity in the nasal epithelium. Basal SCC and calcium-dependent chloride secretion were significantly reduced, whereas CFTR-dependent chloride secretion was increased to normal values. Amiloride-sensitive SCC was reduced by transfection but failed to reach significance. The similarity of murine CF nasal epithelium to that in human CF airways makes the model valuable for gene therapy studies.

Animals↗
PubMed1995

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.

Animals↗
PubMed1995

Generation and characterization of a delta F508 cystic fibrosis mouse model.

We have generated mice carrying the most common mutation in cystic fibrosis (CF), delta F508, within the cystic fibrosis (Cftr) gene. Mutant animals show pathological and electrophysiological changes consistent with a CF phenotype. delta F508-/- mice die from peritonitis and show deficiencies in cAMP-activated electrogenic Cl- transport. These mice produce delta F508 transcripts and show the temperature-dependent trafficking defect first described for the human delta F508 CFTR protein. A functional CFTR Cl- channel not demonstrated by null CF mice or present at 37 degrees C was detected following incubation of epithelial cells at 27 degrees C. Thus, these mice are an accurate delta F508 model and will be valuable for testing drugs aimed at overcoming the delta F508 trafficking defect.

Animals↗
PubMed1994

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

Electrogenic ion transport in the isolated colonic 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 forskolin 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.

Amiloride↗
PubMed1994

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.

Amino Acid Sequence↗
PubMed1994

Kinin-stimulated chloride secretion in mouse colon requires the participation of CFTR chloride channels.

The effect of lysylbradykinin on electrogenic chloride secretion in the epithelium of the mouse colon has been investigated. The peptide was active only when applied to the basolateral surface and its effects were inhibited by the B2 receptor antagonist, Hoe 140, also applied to the same surface. The chloride channel blocker, niflumic acid, also inhibited the response to kinin when added apically. Cyclo-oxygenase inhibition with piroxicam attenuated the responses to kinin, indicating involvement of prostaglandins in the responses. It is concluded that lysylbradykinin increases chloride secretion by acting via B2 receptors and, as with other tissues, brings about secretion through the agency of multiple messengers. In colonic epithelia from cystic fibrosis (CF) mice lysylbradykinin was without effect, suggesting that the final effector process involves apically located cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels.

Animals↗
PubMed1993

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.

Animals↗
PubMed1993

Sequence-selective binding of amiloride to DNA.

Nuclease footprinting techniques have been employed to investigate the interaction between the diuretic drug amiloride, a sodium channel blocker with potential therapeutic use in the treatment of cystic fibrosis, and three DNA fragments of defined sequence. Using either DNAse I or micrococcal nuclease as probes, an unusual pattern of sequence-selective recognition of DNA has been detected. Amiloride binds selectively to sites rich in adenine and thymine residues, frequently with an apparent preference for 5'-TpX-3' steps, and discriminates strongly against GC-rich sequences which are sometimes cut more readily in the presence of the drug compared to the control. A detailed comparison with the actions of known selective DNA-binding antibiotics and drugs reveals a unique pattern of binding sites, different from those of typical intercalators on the one hand and those of minor groove-binders on the other. Amiloride is believed to adopt a pH-dependent tricyclic hydrogen-bonded conformation in solution which allows it to intercalate into DNA; consistent with this belief, we find that the footprinting pattern largely disappears at pH values above the pKa. Preliminary studies with three amiloride analogues have indicated the importance of two functional groups in the recognition of DNA. The possible relevance of selective DNA binding to activity in vivo is considered.

Amiloride↗
PubMed1993

Production of a severe cystic fibrosis mutation in mice by gene targeting.

We have used gene targeting in embryonic stem cells to introduce an HPRT mini-gene into the coding sequence of the murine cystic fibrosis gene (cftr). This insertion introduces a termination codon in frame with the cftr coding sequence to terminate prematurely the CFTR protein within the first nucleotide binding domain. Animals homozygous for the cftr disruption fail to thrive and display a range of symptoms including meconium ileus, distal intestinal obstructions, gastrointestinal mucus accumulation and blockage of pancreatic ducts. The animals also show lacrimal gland pathology. Tracheal and caecal transepithelial current measurements demonstrate the lack of a cAMP activatable Cl- channel. These animals will prove useful for the evaluation of new therapeutic drugs and gene therapy strategies.

Animals↗