Molecular and cellular biology of cystic fibrosis.

Molec. Aspects Meal. Vol. 12, pp. 1-81, 1991 Printed in Great Britain. All rights reserved.

0098-2997/91 $0.00 + .50 ©1990 Pergamon Press plc.

MOLECULAR AND CELLULAR BIOLOGY OF CYSTIC FIBROSIS M.A. McPherson and R.L. Dormer Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff CF4 4XN, U.K.

Contents ABBREVIATIONS Chapter 1. INTRODUCTION History of CF Clinical Features Management of CF New Era for CF Chapter 2.

IDENTIFICATION OF THE CF GENE How the Search Began Discovery of Linkage to CF The Met-J3. I 1 Region Containing the CF Gene Cloning and Identification of the CF Gene Chromosome walks and jumps Identification of gene sequences Tissue expression The CF gene Genetic and Clinical Correlations Implications for Genetic Screening and Diagnosis

8 8 9 10 11 11 12 13 13 14 15

Chapter 3.

EPITHELIA AFFECTED IN CF Structure and Function Exocrine glands Airways and intestinal tract Mechanisms of Fluid Secretion Apical membrane C1- channels Bicarbonate secretion Electrolyte Reabsorption Mechanism of Protein Secretion

17 17 17 18 19 22 24 25 26

Contents

2 Chapter 4.

MECHANISMS REGULATING EPITHELIAL CELL FUNCTION Regulated Events in Epithelial Cells Extracellular Stimuli Stimulus-Response Coupling Second messenger formation Criteria for involvement Mechanism of action

30 30 30 31 32 35 37

Chapter 5.

ALTERED REGULATION IN CF EPITHELIAL CELLS Electrolyte Abnormalities Altered Responses in CF Patients Defective Regulation in CF Cells Salivary glands Sweat glands Airways cells Intestine Mechanisms Causing Defect in Regulation Direct measurement of C1- channels Protein phosphorylation

40 40 41 41 41 42 42 43 43 43 45

Chapter 6.

RELATION OF THE CF GENE TO EPITHELIAL DYSFUNCTION Structure of CFTR Determining Function of CFTR The Superfamily of ATP-dependent Transporters Ion-transporting ATPases Is CFTR an Ion Channel ? Summary of Hypotheses

47 48 48 49 51 51 53

Chapter 7.

CF MODELS Pharmacological Genetic Cultured Cells

56 56 57 58

Chapter 8.

SUMMARY AND PERSPECTIVES Implications of CF Gene Discovery Improvements in Management of CF Prospects for Development of New Treatments Gene therapy Pharmacological approaches

61 61 61 62 62 63

ACKNOWLEDGEMENTS

64

REFERENCES

64

Abbreviations 9AC BAFFA bp CCK cDNA CFTR Cyclic AMP Cyclic GMP AF508 ER G-protein GABA HSV-thyk I

IBMX Ins(1,4,5)P 3 Ins(1,3,4,5)P 4 IRP kb met NPPB ORF PCR PI PIP2 pS PS RFLP SRP V

Anthracene-9-carboxylic acid 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid base pair Cholecystokinin Complementary DNA Cystic fibrosis transmembrane conductance regulator protein Adenosine-3',5'cyclic monophosphate Guanosine-3'5'-cyclic monophosphate Deletion of phenylalanine at position 508 Endoplasmic reticulum GTP-binding protein y-amino butyric acid Thymidine kinase gene of herpes simplex virus Current 3-isobutyl- l-methylxanthine myo-inositol 1,4,5-trisphosphate myo-inositol 1,3,4,5-tetrakisphosphate Gene coding for int- 1 related protein kilobase met oncogene probe 5-nitro-2-(3-phenylpropylamino)benzoic acid Open reading frame Polymerase chain reaction Pancreatic insufficiency Phosphatidylinositol 4,5-bisphosphate picoSiemens Pancreatic sufficiency Restriction fragment length polymorphism Signal recognition particle Voltage

Chapter I

Introduction

Cystic fibrosis or CF as the disease is generally known, is a very common, life-threatening genetic disease in Caucasian populations. It is inherited as an autosomal recessive trait and approximately 1 in 20 people in the U.K. are estimated to be carriers of the CF gene. As the CF inheritance pattern shows (Fig. 1.1) two carriers marrying have a 1 in 4 chance of producing an affected child.

Parents, obligate heterozygotes

@

I

I

I

C.ro.,osome car in0:

~ [~ Homozygote I Normal Heterozygote~ I I I CF

BI

I

,

I N J

Normal gene I CFgene

Cli!i~l (carriers)~

symptoms

Asymptomatic Fig. 1.1. Pattern of inheritance of CF.

Until September 1989, the basic genetic defect in CF was unknown. However, after a search lasting almost a decade, the CF gene has been identified (Rommens et al., 1989; Riordan et al., 1989; Kerem et al., 1989). In conjunction with increasing knowledge of the physiology of affected epithelial cells in CF (McPherson and Dormer, 1987, 1988) this affords a real possibility that the underlying biochemical mechanisms leading to the symptoms of CF will be discovered and a rational way of intervetation to correct the abnormality devised.

Molecular Basis of Cystic Fibrosis

5

History o! CF CF was perhaps first referred to in an early Swiss-German folklore rhyme (Schmidt, 1729) which gives a poor prognosis for the child which tastes salty. This suggests that the gene responsible for causing CF first mutated several hundred years ago. It was not until the 1930s that CF was first accurately described (Fanconi et al., 1936; Andersen, 1938), when it was recognised as a definite syndrome having symptoms of both 'cystic pancreas fibromatosis and bronchiectasis'. Andersen (1938) called the disease cystic fibrosis of the pancreas because of the fibrotic cysts seen in postmortem pancreatic material of CF patients, a name which is commonly shortened to cystic fibrosis or CF. It was later realised that CF is a disease of the exocrine glands, with inspissations in the mucus secretions and this led to the alternative term mucoviscidosis (Farber, 1945). Besides pancreatic and lung involvement, the sweat of CF patients was shown to contain elevated sodium chloride concentrations (di Sant Agnese et al., 1953). This discovery followed a New York heatwave when it was reported that several CF patients were admitted to hospital with heat prostration (Kessler and Andersen, 1951). Although elevated sweat electrolytes are not normally clinically relevant, this was shown to be a very useful diagnostic test for CF (Gibson and Cooke, 1959). Indeed, measurement of electrolyte content of pilocarpine-stimulated sweat is still the main diagnostic test for CF ; with the majority of CF children having values of sodium and chloride between 80 and 125mmol/1 (Goodchild and Dodge, 1985). The sweat test must be done in recognised centres to obtain accurate determinations. It is unlikely to be replaced in the near future with a genetic diagnostic test for CF, because of the finding of many mutations within the CF gene (see Chapter 2). However diagnosis of the small numbers of children who have borderline sweat tests can present problems, and in some cases genetic analysis has given an unequivocal diagnosis (Patton et al., 1987).

Clinical Features All of the clinical manifestations of CF arise from the basic defect, which is a mutation in a single gene. The CF gene codes for a single defective (or missing) protein which causes an alteration in function of the exocrine and epithelial cells, resulting in production of abnormal mucous secretions in the lungs and digestive tract (Fig. 1.2). 5CF pancreatic secretions contain precipitates which lead to ductal obstruction and eventually to acinar cell degeneration and extensive fibrosis. This leads to a deficiency of pancreatic enzymes and hence problems of inadequate nourishment. Intestinal obstructions caused by inspissations in the gut secretions can lead to complications such as meconium ileus in the neonate which may require surgery (Goodchild and Dodge, 1985). The main life-threatening clinical problem for the CF patient however is in the lungs and airways which become obstructed with viscous mucus, leading to chronic pulmonary infections. The CF lung has a tendency to become colonised with the pathogen Pseudomonas aeruginosa, which in its mucoid form produces alginate. This is a mucoid polysaccharide which exacerbates the 'sticky' lung environment (Russell and Gacesa, 1988). Lung damage resulting from recurrent bacterial infections is the main cause of death in CF patients. There is considerable variation in the severity of clinical symptoms of CF and prognosis can vary greatly from one individual to another. Some CF individuals have markedly little pancreatic involvement and mild lung disease; whereas in many both the pancreas and lungs are severely affected; in others liver disease is present (Taussig, 1984; Goodchild and Dodge, 1985). Clinical findings therefore suggested that although population studies pointed to a defect in a single gene (Romeo et al., 1985), CF was not due to a single mutation within that gene. Indeed, the recent

6

M.A. McPherson and R. L. Dormer

molecular genetic data has shown that more than one mutation is present in the CF gene (Kerem et al., 1989).

I CF GENE I

CF protein (Altered or missing)

Altered function of epithelial cells

Altered mucous secretions

High sweat salt

Viscous mucus in lungs

Pancreatic duct obstruction

Intestinal obstruction

Obstruction

Fibrosis

Meconium ileus in neonates

Chronic infections

Lack of pancreatic enzymes Fig. 1.2. Clinical features of CF.

However, in terms of understanding the molecular basis of CF, the common feature is a disturbance in mucus and electrolyte secretion from the exocrine glands and secretory epithelial cells (McPherson and Donner, 1988; McPherson and Goodchild, 1988).

Management of CF Current treatments for CF are aimed at improving quality of life and prolonging life-expectancy, however they are not cures for the disease. Most of the CF patients require oral supplementation of pancreatic enzymes, due to inadequate production by the pancreas. The main management programme for the CF lung disease is extensive physiotherapy to remove the viscous mucus and antibiotic therapy to clear bacterial infections. Although continual progress is being made in these areas (see Chapter 8), they eventually become ineffective in clearing the infected secretions and the bacteria become resistant to antibiotics. This results in the extensive lung damage and fibrosis which is the main cause of death in CF patients. Improvements in therapy have been made over the past several years with the advent of new methods of physiotherapy and antibiotics which have increased life-expectancy. The success of the heart-lung transplantation programme in the U.K.


7

(Geddes and Hodson, 1989) is also encouraging, although this will not be applicable for the majority of CF patients (see Chapter 8).

New Era for CF With the identification and sequencing of the CF gene and the protein coded for by the gene (CF gene protein or CFTR, see Chapter 2), CF research has entered a new era. New methods for diagnosis, screening and carrier testing for CF are being developed which will have profound effects on genetic counselling and disease prevention (see Chapter 2). In addition, the discovery that most (approximately 65-70%) of CF chromosomes in N. America and N. Europe have the same genetic mutation (Kerem et al., 1989; Mclntosh et al., 1989) but that many other mutations are present in the population should provide insights into the aetiology of CF and the variability of the disease. However, potentially the most exciting development for CF will come from determination of the function of the CF gene protein CFTR in epithelial cells and the devising of pharmacological intervention or gene or protein replacement therapies as a means of correcting the CF abnormality. The purpose of this review is to focus on how new information about the CF gene relates to current knowledge of the physiology and biochemistry of epithelial cells which are affected in CF; how they function and are regulated (Chapter 3 and 4); which events are altered in CF (Chapter 5) and how this relates to current ideas as to the structure and function of the CF gene protein (Chapter 6). Chapters 7 and 8 discuss new avenues for future research and the prospects for development of new treatments for CF in light of current knowledge. This is expected to be an exciting era for CF research in which the coming together of advances in understanding human epithelial cell physiology over the past decade and knowledge of the genetics of CF perhaps affords the best hope for CF patients for the development of a rational new treatment aimed at curing the disease.

Chapter 2

Identification of the CF Gene

It has long been recognised that CF is inherited as a recessive trait and that this was an autosomal rather than a sex-linked mode of inheritence (Danks et al., 1965). Later, more detailed studies of relatively homogeneous populations stongly suggested that CF was the result of a mutation in a single gene (Romeo et al, 1985). With the advent of molecular genetic techniques for analysing and sequencing DNA and mapping genes on chromosomes, it has been possible to exactly pinpoint the gene responsible for causing CF. The decade of the 1980s has coincided with the search for the CF gene and perhaps appropriately the final identification came at the end of the decade in September 1989, with the publication of three papers in Science (Rommens et al., 1989; Riordan et al., 1989; Kerem et al., 1989). This exciting discovery resulted from a collaboration between the groups of Tsui, Riordan and Collins in Toronto, Canada and Michigan, U.S.A. However, many other researchers have made vital contributions to this goal, in particular Williamson's group in London, U.K. It is surely the challenge of the 1990s to use this information in conjunction with increasing knowledge of the cellular physiology and biochemistry of CF to devise a rational treatment for CF patients.

How the Search Began Unlike disorders of the blood such as sickle cell anaemia and thalassemia, where it was known that an alteration(s) in the protein haemoglobin was responsible for causing these defects, for CF there was no information about the type of protein coded for by the CF gene and no knowledge as to which chromosome the CF gene was on. Out of 23 pairs of chromosomes, only the X and Y chromosomes had been excluded, in contrast to diseases such as Duchenne muscular dystrophy where the gene was known to be carried on the X chromosome. So to determine the basic defect in CF by 'reverse genetics' , that is chromosomal localisation and identification of the gene without first knowing its sequence, presented a formidable challenge to molecular geneticists. The genetic search began in the early 1980s, when studies in CF families looked for linkage between defined sections of DNA and CF. This relied on the basis that DNA varies in sequence from one individual to another. This natural variation, which is not deleterious, is known as polymorphism. Characteristic phenotypic traits also exhibit polymorphisms, for example difference in eye colour and blood group. The sequence of bases in DNA gives rise to the amino acid sequence of proteins, therefore it is not surprising that proteins also show polymorphisms. For example, the activity of an enzyme can vary from one individual to another without leading to a severe dysfunction. A deleterious change such as that seen in the haemoglobinopathies and CF is defined as a mutation. Before it was possible to study polymorphisms in DNA, polymorphic protein markers were used to look for linkage to CF, to see whether this would provide a clue as to the


9

location of the CF gene (Goodchild et al., 1976). However the probability of finding linkage with CF with a few protein markers covering only a small portion of the human genome was very low and the studies proved negative. Even using DNA markers or probes, it was five years before a linkage to CF was established. Polymorphisms in DNA were tracked using restriction enzymes, which were discovered in soil bacteria. A large number of different enzymes have been identified and each one cuts DNA into fragments at specific sequence sites in the molecule. Any variation in the sequence of DNA between individuals that causes a change in the specific cleavage site of a restriction enzyme would lead to the production of different fragment lengths of DNA (Fig. 2.1). These are known as restriction fragment length polymorphisms (RFLPs) (Botstein et al., 1980) and are recognised following cleavage of DNA with a restriction enzyme by hybridisation to a specific labelled DNA probe. By tracing the different polymorphisms through CF families with more than one affected CF child, it was possible to determine whether a segment of DNA (probe or marker) was linked to CF. Figure 2.1 shows the principle of using RFLPs in linkage analysis through families. The closer the marker was to the CF locus, the more likely it would be to segregate with CF; statistical techniques were used to determine the probability of linkage. Using many anonymous DNA probes on different chromosomes (Tsui et al., 1985) and also gene sequences for candidate proteins for the CF defect, which had been shown to have altered activity in CF cells (Scambler et al., 1987) a large percentage (approximately 40 %) of the human genome was excluded for CF before linkage was discovered.

Z• 2,2

Allele 1 ~ 1,2

Allele 2 Restriction enzyme site

Fig. 2.]. Use of RFLPs in linkage analysis and prenatal diagnosis. Allele l, no resUicdon enzyme site

- larger DNA fragment on gel; Allele 2, restriction enzyme cleavage site - smaller DNA fragment on gel. The affected child (0) has inherited allele 2 from each parent and hence a foetus (A) possessing allele 1 must be either an unaffected heterozygote(as shown) or homozygote(allele 1 only).

Discoveryof Linkageto CF Perhaps surprisingly, the first linkage to CF came from a biochemical rather than a genetic source. It was shown by Eiberg et al. (1985) that a polymorphic activity of the serum enzyme paraoxonase segregated with CF in family linkage studies. The enzyme has nothing to do with the cause of CF; it has an obscure activity in that it hydrolyses paraoxon, a component of weed killer, into pnitrophenol and diethylphosphate. The discovery was not immediately useful for chromosomal localisation of the CF gene, since the gene coding for paraoxonase (PON) had not been assigned to a particular chromosome. In addition, the linkage to CF was not very tight, with a recombination fraction of 10% (Eiberg et al., 1985; Schmiegelow et al., 1986). However the finding of a linkage, however weak, to CF gave a new impetus to the research to track down the CF gene. Spurred on by the news of the biochemical linkage to CF, molecular geneticists intensified their activities, Soon one of two hundred random polymorphic DNA probes (DOCR1-917 or D7S 15) was

10


found to be linked to both CF and PON (Tsui et al., 1985). The genetic distance from CF was approximately 15 centimorgans (cM), as estimated by a recombination fraction of 15%, which corresponds to approximately 15 million base pairs of DNA. The DNA marker was localised to chromosome 7 using a panel of rodent-human cell lines, which contained different human chromosome constituents. It was found that the probe DOCR1-917 hybridised to DNA extracted from cell lines containing human chromosome 7, but not to those cell lines in which this chromosome was absent. (Knowhon et al., 1985). Several other probes on chromosome 7 were soon shown to be linked to CF; two of which - the met oncogene probe (met) (White et al., 1985) and the anonymous DNA sequence J3.11 (D7S8) (Wainwright et al., 1985) were much closer to the CF locus. Studies on hybrid cell lines containing partial deletions of chromosome 7 indicated that the CF locus was located on the middle of the long arm of chromosome 7 (Wainwright et al., 1985). On the basis of collaborative studies which showed a frequency of genetic recombinations between the met and J3.11 probes and CF of approximately 1%, it was estimated that the probes were approximately l c M or 1 million base pairs from the CF locus (Beaudet et al., 1986; Lathrop et al., 1988). The new probes were close enough to be useful in prenatal diagnosis in the majority (approximately 80%) of families with an already affected CF child (Farrall et al., 1986) and could be used for carrier detection in some families (see later section). The order of the probes on chromosome 7 shown to be linked to CF (Zengerling et al., 1987) is shown in Fig. 2.2.

Short arm

D7S15 22 1 ~-~

ICOLIA IMET

31"{3~k~ 1J3.11

COMA 2 D7S15 PON J3.11

TCRB Fig. 2.2. Order of probes on chromosome 7. Adapted from Zengerling et al. (1987) to show the extent of genetic mapping data at that time. The left hand diagram depicts chromosome 7 with vertical lines showing the extent of the regions where each probe is located. The right hand vertical line shows the long arm with horizontal lines marking the most probable location of each probe and the CF locus.

The Met-J3.11 Region Containing the OF Gene The discovery that the met oncogene probe (met) and J3.11 (D7S8) were within two million base pairs of the CF gene and that these markers flanked the CF locus (White et al., 1985; Wainwright et al., 1985; Beaudet et al., 1986) was undoubtedly a major step towards cloning and identification of the CF gene. The distance between the probes, which had been estimated on the basis of genetic recombination, was more accurately determined as 1.5 +- 0.5 x 106 base pairs by physical mapping of the region using pulsed-field gel electrophoresis (Drum et al., 1988; Poustka et al., 1988), a


11

technique which enables separation of larger (>250kb) DNA fragments than conventional agarose electrophoresis. Alternative strategies were then adopted to clone the CF gene. Theoretically this would be possible by cloning all the DNA sequences between the flanking markers. However, this technique, known as chromosome walking, which involves isolating overlapping DNA sequences and monitoring the progress of the walk by detection of restriction sites and measuring the degree of linkage disequilibrium between the polymorphic markers and the CF gene, is a very difficult and timeconsuming task, particularly as some regions of DNA are unclonable. So because of the genetic distance involved, it was necessary to look for additional markers between met and J3.11, which would be closer to the CF gene. One approach was to use the transforming properties of the met oncogene to isolate the DNA region containing both met and J3.11 and by inference the CF gene. DNA from human cells containing the activated met oncogene was introduced into an untransformed mouse cell line. The mouse cells which incorporated segments of chromosome 7 containing the met oncogene grew as transformed clones, from which DNA was extracted. Clones which hybridised with both met and J3.11 were identified from cosmid libraries prepared from the hybrid cell lines. Providing that no chromosomal rearrangement had occurred, they should contain the CF gene. Using these techniques, a candidate gene was identified (Estivill et al., 1987) by the finding of a region rich in GC residues (HTF island), which is often present at the start of a gene and is characterised by restriction sites for enzymes that cut GC rich regions, such as Not 1. The gene was shown to be between met and J3.11 by physical mapping using pulsed field gel electrophoresis and by an increase in linkage disequilibrium, which occurs when restriction fragment length polymorphisms define different allelic association with CF. Although extremely close to the CF gene, subsequent data showed no sequence differences between the normal and the candidate CF gene; that the expression pattern of the candidate gene was not consistent with the epithelial pathophysiology of CF and that recombinations were observed between the gene and CF (Wainwright et al., 1988). The gene (IRP) which codes for int-1 related protein (Wainwright et al., 1988), was therefore distinct from the CF gene. However, it was apparent that IRP was within one or two genes of the CF gene. Using genetic linkage disequilibrium as a monitor of closeness to the CF gene, new probes were isolated: KM19, in particular, which identifies a Pst 1 polymorphism, has been extremely useful for defining haplotype groups and in prenatal diagnosis in families with an affected CF child (Feldman et al., 1988; Newton et al., 1989). One of the probes isolated was later shown to be part of the CF gene. Another approach, saturation mapping, involved the isolation of random single-copy clones from a chromosome 7 library and mapping them to regions of the chromosome using somatic-cell hybrids containing portions of chromosome 7. By isolating a large number of probes, it was assumed that some of them would map between met and J3.11. Indeed, this technique also provided additional markers between met and J3.11 (D7S122 and D7S340) (Rommens et al., 1988). The new clones provided start points for chromosome walking and enabled investigation of gene sequences between met and J3.11.

Cloning and Identification of the CF Gene Chromosome walks and jumps A collaborative effort between three groups (Rommens et al., 1989; Riordan et al., 1989; Kerem et al., 1989) led to the final identification of the CF gene. Figure 2.3 shows the location of the CF gene between met and J3.11. The starting point to the gene identification was the mapping of the newly discovered markers between met and J3.11. Thus, genetic and pulsed field gel

12


electrophoresis mapping indicated that the order of the four markers was met- D7S340 - D7S122 J3.11 (D7S8) with distance intervals of 500, 10 and 980 kbase pairs respectively (Rommens et al., 1989). The estimate of the met-J3.11 (D7S8) distance agreed well with previous data (Beaudet et al., 1986; Lathrop et al., 1988). A large amount of DNA surrounding the probes was cloned and a 280kb (280,000 base pair) segment of DNA, which genetic data suggested should contain the CF gene, was isolated. A new technique of chromosome jumping (Collins et al., 1987) was crucial in speeding up the search for the gene. This involves the ligation of two ends of a large genomic segment, which allows the cloning of sequences which are a considerable distance apart (50-200kb) on the chromosome. The advantage of this technique is that it is much faster than sequential chromosome walking: a new chromosome walk can be initiated in both directions along the chromosome from the end point of each jump. By using this technique, it was also possible to jump over unclonable regions of DNA often found in the mammalian genome, which would end a chromosome walk. Since the new probes found (D7S340 and D7S122) were only 10kb apart, they effectively only provided a single starting point for the walks and jumps. The direction of walking and jumping was determined by the identification of rare restriction enzyme cutting sites The finding that a Not 1 site corresponded to the one associated with the IRP gene (Rommens et al., 1989) and that evidence had suggested that CF was located between IRP and J3.11 (D7S8) (Farrall et al., 1988; Kerem et al., 1989) narrowed down the length of DNA to be examined.

D75122

t

KM19

I ,

[, 500

, 120

|

150 a

I 250

j

' 550

!

Distance (Kilobases)

Fig. 2.3. The met-J3.11 region of chromosome 7. I d e n t i f i c a t i o n of g e n e s e q u e n c e s

Many genes have been conserved in evolution; therefore one way of identifying genes is to investigate whether the sequence cross hybridises to sequences of DNA from other species. This approach was used since it is the most straightforward and has been successful in detection of other disease genes. The technique provided evidence for four transcribed sequences in the 280kb segment of DNA; one was outside the IRP-J3.11 interval and another was the IRP gene itself, which had already been excluded from being the CF locus (Wainwright et al., 1988). Although the third sequence showed open reading frames (ORF), no RNA transcripts or cDNA clones were detected with this probe. The fourth sequence was further analysed, in that DNA segments which detected bovine hybridisation signals were used to probe cDNA libraries from affected epithelial cell types in CF. Seven different libraries were screened and one single clone was isolated from a cDNA library made from cultured sweat gland epithelial cells from a normal individual. DNA sequencing showed that this clone contained a potential open reading frame sequence indicative of an expressed sequence (a gene). The probe was shown to hybridise to a 6.5kb RNA transcript from colon carcinoma and nasal polyp cell lines and pancreas; suggesting that this is the size of mature mRNA of the putative gene. None of the cDNA clones isolated were this size: twenty had to be isolated to sequence the full length of the transcript, which was thus derived from the sequencing of overlapping cDNA clones. Together the clones spanned a region of approximately 6.1kb and contained an open reading frame (ORF) encoding a protein of 1480 amino acids. For confirmed cDNA clones, corresponding genomic segments were isolated and the exons and exon-intron boundaries were sequenced. Twenty four exons (coding sequences) were initially identified within the gene (Riordan et al., 1989, Fig. 2.4). The data has recently been revised to at least 26 exons; with the finding that exon 6 and exon 17 are split (Tsui et al., 1990).

Molecular Basis of Cystic Fibrosis Tissue

13

expression

The mRNA of the candidate CF gene was found to be of low abundance; in the colon carcinoma cell lines, it was estimated to be only 0.01% of the total mRNA. Transcripts of a similar size to those found in pancreas and cultured colon and nasal cells (6.5kb) were found in lung, colon, cultured sweat gland cells, placenta, liver and parotid gland, but the hybridisation signal was less than that detected in pancreas and nasal polyps, mRNA transcripts were not detected in brain, adrenal gland, skin fibroblast or lymphoblastic cell lines. Thus the expression of the candidate gene coincided with the types of epithelial cell which are known to be affected in CF.

t

250,000 base pairs

VI Vlll IX

.

,,

'

' '

,,,

x,x,,

X~q XVU XlII X V / /

XXlU

x.q E.x...

II

II

,,,,

1

'

'

'"

OENE (DNA)

Transcription and RNA splicing 6.500 I~se pairs I

I Translation I

t 1

PROTEIN

1,480 amino acids

Fig. 2.4. The CF gene. Exons (regions which code for protein) are denoted as vertical bars and Roman numerals (I-V). Intervening (non-coding) sequences, introns, are transcribed into nuclear RNA but spliced out to form the cytoplasmic messenger RNA which is lranslated into protein. No significant differences in amount or size of mRNA transcripts were seen between control and CF tissues. This suggests that the CF gene mutation(s) is a subtle change, resulting in production of a protein with abnormal function, rather than lack of the protein and that the protein produced does not differ greatly in size from that of the normal protein. However this will only be confirmed when an assay for the CF protein is developed and it will be possible to determine whether any of the CF gene mutations lead to absence of CF protein. The CF gene

The gene identified was shown by genetic and physical mapping to be in the correct location for the CF gene (Rommens et al., 1989). The tissue expression of the gene was also consistent with the known aetiology of CF. However, because no large deletions or rearrangements were detected within the CF gene and no assay for the defective protein coded for by the gene was available, a detailed comparison of normal and CF sequences was required. Indeed this analysis provided compelling evidence that the candidate gene identified is the CF gene. A marked difference in sequence between DNA from normal and CF individuals was the loss of 3 base pairs (CTT) (a 3 bp deletion ) in CF DNA which would result in the absence of one phenylalanine amino acid at position 508 in the protein (AF508) (Fig. 2.5). The deletion is in exon 10 of the CF gene (Fig. 2.4). To exclude the possibility that the sequence difference was a DNA polymorphism tightly linked to CF, the proportion of CF individuals carrying the deletion was determined. Genomic DNA samples (blood samples) were taken from CF patients and their parents.

14

M.A. McPhersonand R. L. Dormer

Normal individuals were excluded because of the high carrier frequency of the CF gene. Parents of CF children were suitable, since they were known to carry one normal and one abnormal chromosome. The specific segment of DNA encompassing the region of the gene containing the 3 bp deletion was amplified using oligonucleotide primers flanking the mutation in a polymerase chain reaction (PCR). This was then hybridised to 32p-labelled oligonucleotide sequences specific for the normal and mutant sequences.

NORMAL

Amino Acids

lie 507

CF

DNA sequences

I A T

A T

C Phe 508

Gly 509

Amino acids

lie 507

T

[° T

T

G

° G

T

T

Gly 508

Fig. 2.5. The (AFs08)mutation. The sequence of bases in DNA coding for the region surrounding Phe at position 508 in CFFR is shown for the normal and CF gene. It was found that 68% of Canadian CF chromosomes had the phenylalanine ( ~ 5 0 8 ) deletion, whereas out of almost 200 normal chromosomes (now several thousand worldwide) analysed, none were shown to have the deletion (Kerem et al., 1989). This was very strong genetic evidence that the AF508 mutation is specific for CF and not simply a neutral polymorphism and that this is the major mutation causing the disease. In addition, no recombination has been detected between the AF508 mutation site and CF in affected families. The initial evidence was greatly strengthened by haplotype analysis, where the AF508 mutation was correlated with extended haplotype analysis based on 23 DNA markers for CF and normal chromosomes (Kerem et al., 1989). Five major groups of normal and CF haplotypes were defined by RFLPs within or adjacent to the CF locus. It was found that the AF508 mutation associated almost exclusively with the most frequent CF haplotype. The finding that AF508 was detected in 89% of CF chromosomes with this haplotype, but that no deletion was found in 14 normal chromosomes within the same haplotype group confirmed that the AF508 mutation is not a neutral polymorphism associated with a particular haplotype.

Genetic and Clinical Correlations The CF phenotype can be subdivided into two groups, those who have pancreatic insufficiency (PI) and require pancreatic enzyme replacement and a minority which are pancreatic sufficient (PS). It


15

was found that most CF-PI patients were homozygous for the AF508 mutation and it has been assumed that this mutation corresponds to a severe allelle (Kerem et al., 1989). Most of the CF-PS patients carried one copy of the AF508 mutation and were therefore compound heterozygotes. Thus it is assumed that CF-PS carries either a single severe allele or two mild alleles. Meconium ileus occurs in approx 5-10 % of new borns with CF. Five out of six patients studied were homozygous for AF508; therefore it was speculated that homozygous AF508 (or equivalent severe mutations) is a prerequisite for development of meconium ileus (Kerem et al., 1989). However, meconium ileus occurs in only a small portion of CF patients, many of which are homozygous AFs08. Differences in severity of pulmonary disease in a pancreatic sufficient (PS) CF family were reported in individuals with different AFs08 genotype (Santis et al., 1990a). In addition, an association between the AF508 mutation and raised energy expenditure in CF patients (as measured by increased basal metabolic rate) has been reported (O'Rawe et al., 1990). However, an extended analysis of genotype and pulmonary disease (Santis et al., 1990b) revealed that the homozygous AF508 genotype was associated with both mild and severe pulmonary disease, as was the compound heterozygous genotype. The finding (Santis et al., 1990b) that one of the alleles defined by a Pst 1 polymorphism using the probe J3.11 was associated with a mild form of CF suggests that gene(s) close to the CF gene influence clinical outcome. Thus, although some relationships between the AF508 deletion and CF phenotype have been noted, absolute correlations have not been found. A greater understanding of how CF genotype determines phenotype should come from the identification of other mutations in the CF gene. A frame-shift mutation predicted to result in production of a severely truncated protein has been found in a mildly affected CF individual, who is a compound heterozygote and might carry a mild CF allele (White et al., 1990). Examples are now being found of CF patients homozygous for mutations predicted to severely disrupt CFTR (or lead to its absence), with relatively mild clinical symptoms (Cutting et al., 1990a). Evidence thus increasingly suggests that the manifestations of CF are determined not only by the type of mutation in the CF gene, but by other genetic and environmental factors.

Implications for Genetic Screening and Diagnosis A major advance towards screening and diagnosis for CF has come from the finding that direct mutation analysis (Fig. 2.6) is possible for the most common CF mutation (Kerem et al., 1989). The ability to amplify a specific region of DNA using PCR has increased the sensitivity of the detection method In CF families carrying the AF508 mutation, it will be possible to offer unambiguous carrier detection and prenatal diagnosis, even in the absence of DNA from an affected child (Lemna et al., 1990). Previously linkage with the closely linked DNA markers could only be used for prenatal diagnosis in families in which DNA was available from an affected child and cartier detection was only possible in some cases (Farrall et al., 1986; Feldman et al., 1988). In families which were uninformative, microvillar enzyme testing could be employed (Brock et al., 1985), but this was not absolute and had to be carried out at a later stage of pregnancy. Detection of carriers in the population also could not be achieved with linked probes; direct analysis of the gene mutation(s) is required. Unfortunately, this is not going to be straightforward for CF, since it is predicted that over a hundred mutations will be found within the CF gene. Currently at least fifty have been identified (Tsui et al., 1990; Cutting et al., 1990a,b; Dean et al., 1990; White et al., 1990); the fact that many are private (i.e. only found in one family) and that genetic analysis has concentrated mainly on one region of the CF gene suggests that large numbers are present. Pilot schemes to detect CF carriers in the population are underway in the U.K., using direct analysis of the AF508 mutation. However, since only 65-70% of CF chromosomes carry this mutation (Kerem et al., 1989; Mclntosh et al., 1989) only approximately two thirds of carriers (3% of the population) will be detected. In terms of diagnosis only up to 50 % of CF homozygote patients without a family history would be accurately diagnosed (Kerem et al., 1989). Thus in many families not carrying the

16

M.A. McPherson and R. L, Dormer

AF508 deletion, linked analysis will still have to be used for prenatal diagnosis (Feldman et aL, 1988; Newton et al., 1989). It will also not be possible to differentiate non carriers from carriers who have a different genetic mutation from the AFs08; however the likelihood of being a cartier with a negative test will be reduced from 1 in 20 to approximately 1 in 60. In southern European populations, the incidence of the AF508 mutation is much lower, where approximately 45% of CF chromosomes carry this mutation (Estivill et al., 1989). Obviously, it is important to identify other mutations in the CF gene before undertaking widespread population screening. Nevertheless, the detection and counselling of previously unidentified CF carriers in the U.K. population in pilot studies; particularly those in families with a CF child and their relatives, should provide valuable information for population screening when the remainder of the mutations in the CF gene (or at least sufficient to be able to detect the vast majority of carriers) have been identified.

Extract DNA Amplify specific region by PCR using primers for normal or CF sequences Run gel Visualise DNA by staining with ethidium bromide or 32 p -labelled probes

difference

~

CF (z~F 508)

Fig. 2.6. Mutation analysis. Illustrated for a CF family; the CF gene denoted by a shaded area. Polymerase chain reaction (PCR) is used to amplify specific regions of the gene and the difference in size detected on agarose gels as depicted. Now that the CF gene has been found, it is imperative to determine the function of the protein coded for by the gene. This will be possible because of advances in understanding the normal physiology and regulation of the epithelial cell types affected in CF, which is discussed in Chapters 3 and 4.

Chapter 3

Epithelia Affected in CF

Structure and Function The epithelia whose physiological function has been shown to be affected in CF are the exocrine glands (pancreas, salivary and eccrine sweat glands) and the epithelia lining the airways and intestinal tract. A common feature of all these tissues is the presence of secretory and reabsorptive components which together, control the final composition of a macromolecule-containing, fluid secretion which has either a digestive or lubricative and protective function.

Exocrine glands In the salivary and sweat glands, the functions of secretion and reabsorption of fluid and electrolytes are segregated in different cell types: acinar (secretory) and ductal (reabsorptive). The glands are organized as blind-ending tubes in which the proximal ends are comprised of acinar cells and the distal tubes are formed by duct cells (Fig. 3.1). The structure of the pancreas is essentially similar, but both acinar and duct cells secrete fluid. The primary secretion of the acinar cells of the pancreas, salivary and sweat glands is a protein-containing isotonic fluid, which is modified as it passes down the gland duct, either by reabsorption of electrolytes to give a final hypotonic fluid as in the saliva and sweat, or by secretion of a second fluid resulting in an isotonic secretion as in the pancreas. The salivary glands and the pancreas are more complex in structure than the sweat gland with acinar cells forming clusters around the closed end with their lumens leading directly into the duct system. The acini also contain centroacinar cells which are thought to contribute to the primary fluid secretion. The duct systems are highly branched with intralobular ducts leading into larger interlobular ducts which ultimately lead to a common duct emptying into the mouth or duodenum respectively. Salivary and pancreatic ducts contain some secretory cells; in particular, mucussecreting cells occur in both gland-types and the submandibular gland intralobular ducts contain granular cells which secrete serous proteins. The sweat gland is a single unbranched tube with the secretory cells forming a coiled tubule leading to the reabsorbtive duct; both parts of the gland consist of a double layer of cells (see Fig. 3.1 ). Sweat contains very low amounts of protein and mucopolysaccharides which are thought to be secreted by the 'dark' cells (Kurosumi et al., 1984). Pancreas and salivary glands secrete large quantities of digestive enzymes (acinar cells) or mucins (acinar cells in submandibular and sublingual salivary glands and mucous glands in the ducts).

17

18

M . A . McPherson and R. L. Dormer

Acinar cells (secretory) B

cn

I O O

co

i O

ee

Fig. 3.1. Structural organisation of the exocrine glands.

Airways and intestinal tract The airways and intestinal tract have a different organization (Fig. 3.2) in that the surface epithelium consists of a number of different cell types which are secretory or reabsorptive or may be capable of both processes. In addition, both tracts have a sub-surface (submucosal) layer which contains secretory glands. In the tracheobronchial region of the airways the surface consists predominantly of ciliated epithelial cells interspersed with mucus-secreting goblet cells (Fig 3.2). The submucosal secretory glands have a tube-like structure, similar to the other exocrine glands (see above), which serves to convey their mucous secretion onto the surface of the epithelium. Both goblet ceils and submucosal glands probably contribute to formation of the mucus layer which overlies the airways surface (Kilbum, 1968). The mucus layer consists of a viscous gel 1-2 ~m thick, under which is a more watery (periciliary) layer in which the cilia of the surface epithelial cells can beat and by interaction with the mucus layer, propel it from the distal to the proximal regions. The functions of the mucus gel are twofold: to trap foreign particles, including bacteria, so that they are transported to the mouth and swallowed and to protect the airways surface from dehydration. The surface epithelial


19

cells are capable of both secretion and absorption of fluid (Welsh, 1986a) which must be balanced to maintain the critical thickness of the periciliary layer (Nadel et al., 1979). However, owing to the large decrease in surface area between the distal and proximal airways, it has been argued that the surface epithelial cells of the bronchi primarily absorb fluid (Kilburn, 1968) and this has been confirmed by ion flux studies on excised human bronchi (Knowles et al., 1984). In addition to its function of digestion and absorption of nutrients, the intestinal tract is required to absorb approximately 8 litres of fluid per day in a normal individual (Binder, 1989). The epithelium of the small intestine is organized to maximise its surface area for absorption by being folded into villi (Fig. 3.2) and by each surface epithelial cell possessing numerous microvilli on its apical surface. At the base of the villi are the crypts which contain four different secretory cell types (Trier and Winter, 1989): endocrine cells which secrete gastrointestinal hormones; goblet cells whose mucus secretion is thought to protect and lubricate the intestinal epithelium; Paneth cells which secrete proteins whose nature and function is not clear and undifferentiated epithelial cells which contribute to fluid and electrolyte secretion by the intestine. The undifferentiated cells undergo mitosis in order to constantly renew the villus epithelium and therefore, during their migration from crypt to villus, differentiate to become absorbtive cells. The submucosal layer of the duodenum contains Brunner's glands which are similar in structure to the exocrine glands. They contain both mucous and serous acinar cells and secrete a fluid containing proteins and bicarbonate into the intestinal lumen. The bicarbonate secretion from these glands and probably, more importantly, from the surface epithelial cells is thought to prevent damage to the epithelial surface by gastric acid entering the duodenum (Flemstrom and Garner, 1982).

Mechanisms of Fluid Secretion The secretion of fluid from epithelia depends upon the ability of the secretory cells to transport electrolytes into the ductal lumen, allowing water to follow by osmosis. The predominant electrolytes in the primary secretions are either NaC1 (exocrine acinar cells, submucosal glands, airways, jejunal and ileal surface epithelial cells) or NaHCO 3 (pancreatic duct and duodenal epithelial cells). In both cases, secretion occurs by primary translocation of the anion from the basolateral to the luminal side of the cell; this creates a potential difference across the epithelium (ductal lumen negative) which drives cations into the lumen, usually via a paracellular route (Fig. 3.3). The mechanism of CI- secretion (Fig. 3.3A) is the best understood and is therefore discussed first, with the proposed mechanism for HCO3-, which is partly a refinement of the C1- secretion model, discussed later. In all cell types the energy for secretion is derived from the Na+,K+-ATPase, located in the basolateral membrane, which pumps 3 Na ÷ ions out of the cell in exchange for 2 K ÷ ions, thereby generating an inwardly-directed electrochemical Na ÷ gradient. Detection of binding of [3H]ouabain (the specific inhibitor of the Na+,K+-ATPase) by autoradiography or enzyme histochemistry, has been used to show the basolateral location of the ATPase in the pancreas (Hootman et aL, 1986), monkey palm eccrine sweat gland (Sato et al., 1988), dog trachea (Widdicombe et al., 1979) and rabbit intestine (Stirling, 1972). In addition, ouabain added to the basolateral side of the cell, has been shown to inhibit secretion from rat pancreatic acinar and duct cells (Evans et al., 1986; Novak and Greger, 1988a), submandibular acinar cells (Martinez and Cassity, 1984), monkey eccrine sweat glands (Sato et al., 1988) and dog tracheal cells (A1-Bazzaz and AI-Aqwati, 1979). Figure 3.3A shows the current model, first proposed by Silva et al. (1977), to explain the mechanism by which CI- is secreted by epithelial cells. Essentially, the cells possess a cotransporter on the basolateral membrane which couples Na + entry down its electrochemical gradient to C1- and K + in the ratio 1Na+:IK÷:2CI and, in the apical membrane, a CI" conductive channel. The activity of the cotransporter results in accumulation of intracellular CI- above its

20

M.A. McPhersonand R. L. Dormer

equilibrium potential such that, when the CI- channel is opened, electrogenic secretion of CI" occurs down the favourable electrochemical gradient.

A Gobletcells

Ciliatedepithelialcells(secretory and reabeorptive)

(secret°ry) F - - - ~

.~

(secretory) B Absorptive cells

Crypt

secretoryc e ~

~s /

Fig. 3.2. Structural organisation of the airways and intestinal epithelium. Evidence f o r involvement of the Na+,K+,2CI - cotransporter has been obtained by demonstration that C1- secretion is dependent on Na ÷ and CI- and inhibited by inhibitors such as furosemide and bumetanide in rat pancreas (Seow et al., 1986), rat and rabbit submandibular (Mardnez and Cassity, 1984,1985a), monkey palm eccrine sweat glands (Sato et al., 1988) and dog trachea (Welsh, 1983; Widdicombe et al., 1983). Direct measurements of intracellular CI" activity, using ion-selective microelectrodes, have demonstrated accumulation of C1- above its equilibrium potential in the

Molecular Basis of Cystic Fibrosis 21 mouse pancreas (O'Doherty and Stark, 1983), rabbit submandibu]argland (Lau and Case, 1988) and dog trachea (Welsh, 1983), giving additional support for the model.

A

t /

I

f

.~ Na +

~Na+~--~ T ~ K+

Na + ~ 2CI- "-I

=- CI

K+

K+ _~ --~L) mUenminbra/ne

Basolateral l membrane f

B L

_/ ---~ Na +

I

Na +

I i

K+..~

I

_

CO2+ H20 I

~ /

1

~ ClI I

/

H2C93 cr~-J~-H*--

. H O;

K+'~

,,

- HCO

J r

Fig. 3.3. Mechanisms of fluid secretion. A. Sodium chloride secretion. The epithelial cell is depicted with basolateral Na+/K+-ATPase (circle) and cotransporter (rectangle); basolateral and apical membrane ion channels (-II-). B. Sodium bicarbonate secretion. Symbols as in A except filled circles denote exchangers. The model requires that there must be at least one additional channel or exchanger to allow secretion to be maintained. Thus, in the model for NaCI secretion, K ÷ would accumulate in the cell

22


and depolarize the basolateral membrane thereby decreasing the electrical driving force for CIsecretion (see Petersen, 1986 for a review). Using patch clamp techniques (described in the next section), Ca2+-activated K + channels have been demonstrated on the basolateral membrane of many secretory epithelial cells including human and pig pancreas, human and rodent salivary glands (see Petersen, 1986), human and dog tracheal and nasal epithelial cells (Welsh, 1986a; Welsh and Liedtke, 1986) and rat and rabbit enterocytes (Sepulveda and Mann, 1985; Morris et al., 1986). Evidence suggests that the channels are opened on stimulation of the cell (see Chapter 4) and that this allows K + to recycle across the membrane (Fig 3.3A).

Apical membrane CI- channels The model of fluid secretion is dependent on the presence of a C1- channel in the apical membrane. Since C1- impermeability has been shown to be a feature of CF cells (see Chapter 5), investigators have focussed studies on the channels which conduct C I across the apical membrane of the epithelial cell. The demonstration of an apical membrane C1- conductance has been achieved using a number of approaches (see Gogelein, 1988 for a more detailed review). In dog and human tracheal cells and human nasal epithelial cells, intracellular microelectrodes have been used to show that stimulation of secretion causes depolarization and a decrease in fractional resistance of the apical membane which is consistent with increased C I conductance across the membrane (Welsh, 1986a; Widdicombe et al., 1985). Studies with isolated apical membrane vesicles from bovine trachea (Langridge-Smith et al., 1984; Dubinsky and Monti, 1986) have also provided evidence for a C1conductance in this membrane. Patch clamp techniques have greatly increased our understanding of C1- channels, since they allow direct recording of single ion channels. This is achieved by bringing a micropipette up against the surface of a cell where it can form a very high resistance (gigaohm) seal which effectively isolates the area of the membrane within the pipette (the patch) from the rest of the cell surface. This allows the measurement of electrogenic ion movements through channels in the patch as minute currents (picoamps), at voltages across the membrane which can be set by the experimenter (see Petersen, 1986 for more detailed information). As shown in Fig. 3.4, recordings can be made in various modes: a) the 'cell-attached' mode, where the patch remains in place in the intact cell membrane so that the opening and closing of channels in response to external stimuli can be monitored; b) the patch can be ripped off the cell by pulling the patch pipette away and then placing the pipette tip containing the patch in a bath containing physiological medium. This is the 'excised, inside-out' patch which allows putative regulators of channel activity to be added to the bath, giving them direct access to the cytoplasmic face of the membrane; c) the membrane patch can be ruptured with the pipette sealed to the otherwise intact cell. This allows 'whole-cell' recording of the major ion currents to be made while the cell is stimulated to secrete . Addition, of agents to the pipette solution also allows manipulation of the intracellular environment. Several different types of CI- channel have been identified in excised, inside-out membrane patches from epithelial cells, but it is not clear which type of channel is involved in secretion. For example, in human tracheal cells, Frizzell et al. (1986) described two channels whereas Welsh (1986b) described only one. Channels were defined mainly by their conductance properties and anion selectivity. Conductance is independent of the voltage imposed across the membrane when the current-voltage (I/V) relationship is linear as in the 20pS conductance channel in human tracheal cells (Frizzell et al., 1986). However, the other CI- channel described in human airways (Frizzell et al., 1986, Welsh, 1986b) does not have a linear I/V relationship, but has properties of outward rectification (i.e. more current flows in the outward direction than in the inward at an imposed positive voltage). Outwardly rectifying CI- channels have also been described in human nasal epithelial (Kunzelmann et al., 1989), sweat coil secretory cells (Krouse et al., 1989) and in two

MolecularBasisof CysticFibrosis

23

colonic tumour cell lines: HT29 (Hayslett et al., 1987) and T84 (Halm et al., 1988). For this type of channel the conductance is defined at a given voltage or conductance values obtained over a range of voltages from negative to positive are given. Thus the outward rectifying channel conductance in human tracheal cells was 25-50pS (Welsh, 1986; Frizzell et al., 1986), when no voltage was imposed across the patches. A channel of similar conductance was seen in colonic T84 cells (50pS) (Halm et al., 1988), nasal epithelial cells (47-74pS) (Kunzelman et al., 1989) and sweat coil (2441pS) (Krouse et al., 1989). Three other channels with linear I/V relationships and conductances of 10, 18 and >200 pS were also seen in sweat coil cells.

Micropipette

__.~.'.:iIiii.:.i

Ml~t~hbrane . ~

CELL-ATTACHED

Cell

EXCISED.INSIDE-OUT

WHOLE CELL

Fig. 3.4. Modes of recording ion channcls in membrane using the patch clamp technique. The outwardly rectifying C1- channel in tracheal and T84 cells had a greater conductance for I- and Br- than for CI" (Frizzell et al., 1986; Welsh, 1986b; Halm et al., 1988) whereas in nasal and HT29 colonic cells all three anions were carried equally (Hayslett et al., 1987; Kunzelmann et al., 1989). Thus, although all C1--secreting epithelia studied to date have been found to possess an outwardly rectifying C1- channel in inside-out membrane patches, it is not clear whether this is the same channel in each cell type, nor whether it is involved in fluid secretion. This may in part reflect differences in (a) cell culture techniques; thus where several channels have been identified, it is not known whether they are on different cell types in the cell culture population and (b) temperature: patch clamp data obtained at physiological (37"C) rather than room (21"C) temperature (Hayslett et al., 1987; Kunzelmann et al., 1989; Welsh et al., 1989) has revealed differences in channel activity. In addition, C I channels recorded in the 'whole-cell' configuration were shown to have different properties depending on the type of stimulus used to activate the cell (Cliff et al., 1990). Whether this reflects the opening of a single type of C1- channel showing different properties depending on the stimulus or opening of distinct C1- channels by different regulators remains to be established. These differences have revealed further insights into how CI" channels are regulated and the abnormality seen in CF cells and this is discussed in Chapters 4 and 5. The most direct evidence that CI- channels are involved in secretion is that activators of secretion increase the open-state probability of channels in cell-attached membrane patches in tracheal cells

24


(Frizzell et al., 1986; Welsh, 1986b) and that inhibitors of secretion such as 9-AC and NPPB inhibit the outward rectifying C1- channel in excised membrane patches (Welsh, 1986b; Kunzelmann et al., 1989).

Bicarbonate secretion Mechanisms regulating HCO 3- secretion are less well understood than those of C1- secretion, however, understanding the links between these two processes is important for understanding the CF abnormality, since evidence indicates that both are abnormal in CF (see Chapter 5). Micropuncture studies in rabbit pancreas (Swanson and Solomon, 1975) and electrophysiological measurements in isolated perfused rat pancreatic ducts (Novak and Greger, 1988a), have suggested that the basolateral membrane contains a ouabain-sensitive Na+,K+-ATPase and an amiloride sensitive Na+/H + exchanger but no Na÷,K+,2C1" cotransporter (Fig. 3.3B). However, bicarbonate secretion is dependent on the presence of Na + (Swanson and Solomon, 1975; Kuijpers et al., 1989a) and CI- (Case et al., 1979; Kanno and Yamamoto, 1977) and is inhibited by high concentrations of furosemide or bumetanide and by SITS (Kuijpers et al., 1984). This has led to the proposal of a Na+-dependent, CI-/HCO 3- exchanger in the basolateral membrane of the rabbit pancreatic duct cell, leading to accumulation of intracellular HCO 3- and its efflux across the luminal membrane down a concentration gradient (Kuijpers et al., 1989b). However, electrophysiological measurements in isolated perfused ducts (Novak and Greger, 1988b) from rat pancreas, have suggested that the C1-/HCO 3- exchanger is in the luminal membrane. A role for apical membrane CI- channels in HCO 3- secretion has been postulated, since CI- channels have been demonstrated by patch-clamping on rat and human pancreatic duct cells (Gray et al., 1988, 1989). The most common channel observed had a smaller conductance (4-7pS) than the outwardly rectifying channel described in human airway epithelial cells (see above). It also showed little outward rectification, unlike a second channel, seen much less often on the duct cell, which had similar properties to the 25-50pS channel of tracheal cells (Welsh, 1986b; Frizzell et al., 1986). It has been suggested that the small C I channel is involved in HCO 3 secretion on the basis that the physiological secretagogue, secretin, increased its open - state probability and because the larger channel was so rarely found on the cell (Gray et al., 1989). Movement through an apical membrane CI" channel has been suggested as the mechanism by which rabbit submandibular acinar cells secrete HCO 3- (Case et al., 1989). The calculated permeability ratio of HCO3-:C1- of 0.6 would be sufficient to explain the low amount of HCO 3- in saliva but is unlikely to be the pathway for HCO 3secretion in pancreatic duct cells. These findings, together with electrophysiological data (Novak and Greger, 1988b) suggesting a luminal-membrane CI-/HCO 3- exchanger, have led to the model shown in Fig. 3.3B, in which intracellular HCO 3- derived from CO 2 would be secreted in exchange for luminal CI- which is recycled through the CI- channel. A criticism of this as a general model for pancreatic bicarbonate secretion is the requirement for a steady supply of luminal C1- (Novak and Greger, 1988b). which could possibly only be maintained from C1- secreted through the apical membrane channel of acinar cells. Secretin however does not stimulate CI- secretion from acinar cells in all species. Duodenal cells also possess a CI-/HCO 3 exchange mechanism and a CIconductance (Brown et al., 1989) suggesting a similar mechanism in this cell type. In order for NaHCO 3 secretion to be maintained, H + rather than K +, has to be extruded from the cell (Fig. 3.3B) and this has been suggested to occur on the Na+/H + exchanger (Swanson and Solomon, 1975; Novak and Greger, 1988a). Much of the evidence for the current models of electrolyte secretion was initially derived from studies on animal tissues. However, as indicated above, a considerable amount of evidence has accumulated to suggest that human cells have the same basic mechanisms.


25

Electrolyte Reabsorption A basolateral Na+/K+-ATPase, which maintains a low intracellular Na + concentration, is also the driving force for Na + reabsorption by salivary and sweat ducts, airways epithelia and cells of the intestinal tract as shown by inhibition of this process by ouabain (Quinton, 1981). Studies with intracellular microelectrodes in rabbit ileum (Nellans et al., 1974), microperfused human sweat ducts (Reddy and Quinton, 1989a) and nasal epithelium (Cotton et al., 1987) have shown that there is an inward electrochemical driving force for Na + across the apical membrane allowing Na ÷ reabsorption to occur (see Fig. 3.5).

Duct lumen Na +

11"

CI-

.~ Na +

--!

K+~~--']

i

Cl-

I

I

I I I I t

Na + w

~

Cl-

~

K+ CI-

r

]

Fig. 3.5. Mechanism of sodium chloride reabsorption in exocrine gland ducts and airways epithelial cells. Symbols are as in Fig. 3.3A. CI- passes through ion channels in both cell membranes or between the cells. Three modes of Na + absorption have been recognised in different regions of the intestinal tract (see Binder, 1989): non-electrolyte-coupled, neutral and electrogenic. Na+ entry into the epithelial cells of the small intestine is coupled to reabsorption of non-electrolytes (sugars, amino acids, peptides) by apical membrane carriers. The osmotic pressure resulting from this movement of molecules pulls water across the epithelium as for secretion. In the jejunum, where the permeability of the paracellular pathway (through the tight junctions) is greater than in the ileum, water absorption is greater and can carry with it, ions and small non-electrolytes by a process termed solvent drag. The greater 'leakiness' of the jejunum is also reflected by its inability to develop as large a transepithelial potential difference as the ileum. Thus, in the ileum not only is less Na ÷ carried by solvent drag but more is recycled to the lumen due to its higher negativity with respect to the serosal side resulting in less net Na + absorption by this mechanism. Early studies, perfusing the human ileum (Turnberg et al., 1970), provided evidence for electroneutral Na ÷ absorption utilizing coupled Na+/H ÷ and CI'/HCO 3" exchangers by a process similar to that for HCO 3" secretion by the pancreatic ducts and duodenum (see Fig 3.3B). The exchangers have been identified by tracer flux studies on isolated membrane vesicles from rat and rabbit ileum and jejunum (Liedtke and Hopfer, 1982; Knickelbein et al., 1985). However, there is also evidence that electroneutral Na + absorption can occur, coupled to CI" on a cotransporter analogous to the basolateral Na+/K÷/2CI - cotransporter of secretory cells. Thus, in rabbit ileum, microelectrode studies suggested that Na ÷ and C1- entry was coupled in a neutral complex (Nellans

26


et al., 1974) and, more recently, a radiotracer study using apical membrane vesicles showed Na +

uptake stimulated by C1- and CI- uptake stimulated by Na +, both being inhibited by furosemide (Fan et al., 1983).

The colon is a higher resistance epithelium than the ileum (Binder, 1989), developes a greater transepithelial potential difference and hence is capable of absorbing Na + against a higher concentration gradient by predominantly transcellular routes. This probably reflects its importance in conservation of salt and water. It possesses no non-electrolyte-linked Na + carriers and absorbs predominantly by electroneutral exchange or by the third mechanism, which is present in all regions of the intestinal tract, electrogenic transport. In this mechanism Na + enters the cell down its electrochemical gradient through a channel which has the common property of being inhibited by ~tM concentrations of amiloride. This type of channel has been demonstrated in colonic, sweat and salivary duct (Quinton, 1981; Knauf et al., 1982; Reddy and Quinton, 1989) and airways epithelial cells (Welsh, 1986a), bronchi (Knowles et al., 1984) and Clara cells (Van Scott et al., 1987). C1- reabsorption appears to proceed passively down a favourable electrical gradient which is generated across the epithelia (Quinton, 1983; Welsh, 1986a; Knowles et al., 1983a; Pedersen et al., 1987; Binder, 1989). In the human sweat duct, evidence from microelectrode studies (Reddy and Quinton 1989a,b) and from the use of C1- channel blockers (Bijman et al., 1987) suggests that C1- enter the cell through an apical membrane channel. The potency of the series of channel blockers used, suggested that this channel is not the same as that thought to be responsible for C1secretion in airways or colonic epithelial cells (Bijman et al., 1987). At lower luminal concentrations of C1- that are found in the sweat duct in vivo, it is possible that the electrochemical driving force would not be sufficient for passive entry of C1- in this way (Reddy and Quinton,1989a) and it has been suggested, on the basis of the effects of luminal bicarbonate on ductal reabsorption, that a C1-/HCO 3- exchanger coupled to a Na+/H + exchanger may have a role in this process (Sato, 1977; Quinton, 1982). Sweat duct cells also possess a K + permeability at the basolateral but not the apical membrane (Bijman and Quinton, 1987) suggesting that K + is recycled across this membrane, as in secretory cells (Fig. 3.4).

Mechanism of Protein Secretion Secretory proteins, such as digestive enzymes and mucins, are stored in a highly concentrated form in membrane-bound granules. They are released by exocytosis, whereby the membrane of the secretory granule fuses with the apical membrane of the cell, allowing the granule contents to be washed into the ducts. The granule membrane is then recaptured and recycled (Palade, 1975). As shown in Fig. 3.6, the translation from messenger RNA, of proteins destined for secretion, begins on cytoplasmic ribosomes. As soon as the N-terminal region of the protein is synthesised it binds a cytoplasmic ribonucleoprotein, termed signal recognition particle (SRP), which arrests further translation until the complex binds to specific receptors in the rough endoplasmic reticulum (ER) membrane. Following dissociation of SRP, translation recommences and the N-terminal sequence, the so-called signal peptide interacts with the ER membrane to allow the growing protein chain to be translocated across the membrane. A proteolytic enzyme (signal peptidase) on the inner face of the ER membrane cleaves the signal peptide and the newly-completed protein is released into the ER lumen. From here the protein is transported to the cis-stacks of the Golgi apparatus and thence to the trans-face during which time glycosylation, which began in the ER, is completed (see Lingappa, 1989 for a review). The protein is then segregated into vacuoles which bud off from the Golgi and, by a process of condensation involving removal of water from the vacuole, mature secretory granules are formed (Palade, 1975; see Fig. 3.6). Proteins destined to be inserted into membranes have a similar mechanism of translation except that, in addition to the N-terminal signal sequence, they contain internal stop-transfer sequences

MolecularBasisof CysticFibrosis Ribosome

mRN_A~

@

,~_.~pSgi nal iptida_~ /

,,0 i-t ha' Membrane

Lumen protein

27

6 @

/

/

Golgstacks i --L0 Condensingvacuole O Maturesecretorygranule( ~

Apicalmembrane

Fig. 3.6. Synthesis and secretion of proteins. A signal sequence is the first segment of the polypeptide chain to be translated and directs translocation across the endoplasmic reticulum (ER) membrane where it is cleaved by the membrane enzyme signal petidase. SRP: signal recognition particle. which hold up segments of the protein in the ER membrane during translation, such that the protein is retained in the membrane and translation of the remaining sequence is completed in the cytoplasm (see Lingappa, 1989 and Fig. 3.7). The protein is then transported to the appropriate site in the c e l l . As shown in Fig. 3.7, this sequence of events produces a surface membrane protein with its N-terminus facing the cell exterior. Many membrane proteins, which might include CFTR (see Chapter 6), have the reverse orientation. They have a signal sequence not at their N-terminus but at an internal site, such that translation is not inten'upted by binding to SRP until the internal signal sequence is synthesised (Fig. 3.7). Thus part of the N-terminal region remains in the cytoplasm during completion of translation and the threading of the protein in and out of the membrane occurs a s a result of stop-transfer sequences.

28


The mechanism of fusion of the granule and apical membranes to cause secretion of proteins is poorly understood. Experiments using model membrane systems have suggested the requirement for an osmotic gradient across the granule membrane, causing it to swell prior to fusion, but there is no satisfactory model to explain the physiological process of exocytosis (Finkelstein e t al., 1986).

ansfersequence ER membrane I sequence

NH 2

CO,~

Fig. 3.7. Mechanisms proposed to retain integral membrane proteins. The left-hand mechanism shows a membrane protein with signal sequence as in Fig. 3.6. The protein is retained in the membrane due to the stop-transfer sequence and has its C-terminus in the cytoplasm. The right-hand mechanism shows a membrane protein with an internal signal sequence and stop-transfer sequence, such that its N-terminus remains in the cytoplasm. The process of translocation of intemal sequences and retention by stop-transfer sequences is repeated for proteins with multiple membrane-spanning segments such as CFTR. Studies on the permeability properties of exocrine secretory granules have suggested an intimate link between protein and electrolyte secretion. It has been shown that isolated granules from both the exocrine pancreas and the parotid gland possess both a CI- and K + conductance (DeLisle and Hopfer, 1986; Gasser e t al., 1988a). The Cl- conductance was shown to be activated in cells stimulated with physiological secretagogues (Gasser e t a/., 1988b). It has been proposed therefore,


29

that when the granule and apical membranes fuse during exocytosis, CI- channels present in the granule membrane will effectively become part of the apical membrane, allowing C1- and thereby water to be secreted through the granule, washing its protein content into the ductal lumen. This may be of particular importance for mucus - secreting cells where the mucins, which are condensed in secretory granules, swell markedly on release from the cell as they hydrate. It has been suggested that the condensed form is retained by shielding of the polyanionic charge of mucins by Ca 2+ in the granules (Verdugo, 1990). Indeed there is a positive correlation between mucin and Ca 2+ content of submandibular gland secretory granules (Muller et al., 1984). Thus, during exocytosis, as water enters the granules Ca 2+ diffuses out of the cell, revealing the polyanionic sites of the mucins which attract water for hydration by osmosis. Therefore, disturbances in ionic content of secretions from CF epithelia such as the observed increase in Ca 2+ (Chemick e~ al., 1961; Davies et al., 1990) may interfere with the degree of hydration of mucins.

Chapter 4

Mechanisms Regulating Epithelial Cell Function

As discussed in the last Chapter, the function of exocrine and epithelial cells is to provide secretions which are a combination of water, electrolytes and proteins, allowing correct digestive, protective and lubricating functions in the gastrointestinal tract and airways. It is therefore necessary for the secretory and reabsorptive processes to be under neuronal and hormonal regulation. That regulation is crucial for providing the correct balance of secretions is evident from disease states such as secretory diarrhea induced by cholera toxin, which permanently switches on adenylyl cyclase in the gut (see Gilman, 1987), causing a continually stimulated rate of fluid secretion. A general view of CF is that the main clinical symptoms are caused by the relative dehydration of the secretions and, in this respect are the 'opposite' of cholera. Indeed it has been suggested that carrying the CF gene has conferred a selective advantage on heterozygotes in protecting against cholera (see Bijman et al., 1988). In view of the cellular abnormality in CF (see Chapter 5) a comparison of the diseases may well be justified and suggests that to understand the CF abnormality, it is necessary to elucidate the mechanisms regulating epithelial secretion.

Regulated Events in Epithelial Cells In terms of controlling fluid secretion and reabsorption, opening of apical membrane C1- channels and basolateral K + channels are likely to be the key regulated events (see model in Chapter 3; Petersen, 1986; Pedersen et al., 1987; McCann and Welsh, 1990). Although there is evidence for regulation of the Na+/K+-ATPase and Na+/K÷/2CI - cotransporter by regulators of secretion (Hootman, 1986; Liedtke, 1989), it is not clear how this contributes to the control process. Acute regulation of protein secretion is at the level of exocytosis, whereby intracellular messengers promote interaction between secretory granules and the apical membrane, leading to fusion and extrusion of the granule contents (DeLisle and Williams, 1986). Evidence that C1- channels are present on secretory granule membranes, which are opened when the cell is stimulated (see Chapter 3), has suggested that protein and fluid secretion are intimately linked and this may be important in understanding the mechanism of the cellular defect in CF cells (McPherson et al., 1986; McPherson and Dormer, 1987). However, the events might be regulated independently since, under nonphysiological stimulatory conditions, evoked by either a pure o~- or 13-adrenergic agonist, stimulation of fluid and protein secretion can be dissociated in salivary acinar cells (Quissell and Barzen, 1980; McPherson and Dormer, 1984).

Extracellular Stimuli Regulation of secretion and reabsorption of electrolytes, fluid and proteins involves the same types of stimuli; the exocrine glands and epithelial cells are innervated by both parasympathetic and 30


31

sympathetic nerves, which release autonomic neurotransmitters. The main neurotransmitters causing secretion are noradrenaline and acetylcholine, with some input from peptidergic transmitters such as VIP and Substance P (for general reviews see Sato, 1977; Hootman, 1986; Matin, 1986; Binder, 1989). Cholinergic and adrenergic stimuli have also been shown to control reabsorption by sweat and salivary duct cells (Schneyer and Thavornthon, 1973; Schwartz and Simpson, 1985; Pedersen et al., 1987). Locally acting agents such as bradykinin and histamine also influence secretion and reabsorption (Matin, 1986; Brayden et al., 1988; Binder, 1989; Boucher et al., 1989). In addition, specific hormones control secretion from the glands: they include the gut hormones secretin and cholecystokinin, which control pancreatic secretion (Petersen, 1984; Hootman, 1986); prostaglandins, which cause fluid secretion from both intestinal and airways epithelial cells (Matin, 1986; Binder, 1989), aldosterone which increases Na + absorption in the colon (Binder, 1989) and adrenaline which acts on airway epithelial cells (Matin, 1986). Fluid secretion from submandibular and parotid acinar cells, sweat coil and airways submucosal glands, is predominantly stimulated by cholinergic and 0~-adrenergic mechanisms (Schneyer et al., 1972; Sato, 1977; Matin, 1986). The pancreas is also stimulated by a cholinergic mechanism in the cephalic phase, but cholecystokinin and secretin are of primary importance in the gastric phase, stimulating the acinar and duct cells respectively (Petersen, 1984). In the pancreas and airways submucosal glands, cholinergic stimulation also results in copius protein secretion (Petersen, 1984; Matin, 1986), whereas in salivary and sweat glands, it is a relatively weak protein secreting stimulus; the primary stimulus in these glands being the ]3-adrenergic action of noradrenaline (Sato, 1977; Hootman, 1986; McPherson and Dormer, 1984). ]3-Adrenergic stimulation increases fluid secretion to a small extent, since it opens apical membrane CI- channels, but not basolateral K + channels (Petersen, 1986; Welsh and Liedtke, 1986) in salivary and airways epithelial cells. Under physiological conditions however, where epithelial cells are exposed simultaneously to or- and ]3adrenergic stimulation by the release of noradrenaline at the cell surface, large quantities of both fluid and protein are secreted. In cultured human sweat duct cells (Pedersen et al., 1987), both cholinergic and ]3-adrenergic stimuli increase C1- reabsorption whereas, in airways cells, ]3-adrenergic stimulation increases both CI- secretion and Na + reabsorption, events which undoubtedly influence the degree of hydration of the airways (Boucher et al., 1986; McCann and Welsh, 1990).

Stimulus-ResponseCoupling The neurotransmitters and hormones bind to specific receptors on the cell surface and transmit their signal instructing the cell to secrete, by increasing the concentration of a second messenger(s) inside the cell. The second messenger activates a specific enzyme, which modifies the function of protein(s) causing the opening of ion channels or fusion of zymogen granules with the cell membrane and release of their contents. As indicated in Fig. 4.1, each stimulator has its own specific receptor but can cause a change in the concentration of more than one messenger; in addition, different stimulators can activate the same messenger pathway. In polarized epithelial cells, the receptors for the major physiological stimulators are located in the basolateral membrane, whereas the second messenger action can be at both the basolateral (K + channels) and apical (CIchannels/exocytosis) membranes. Thus, the intracellular message has to be transmitted across the cell, either by diffusion or by a directed cascade whereby the message is generated at a series of sites throughout the cell. Evidence for second messenger involvement in hormone action came from studies showing that hormones and neurotransmitters do not enter cells but bind to specific receptor proteins on the cell surface. The first demonstration that a second messenger mediated the action of a hormone was the finding that cyclic AMP increased inside the cell in response to the hormones adrenaline and glucagon and that addition of cyclic AMP gave the same response, i.e. activation of glycogen

32


phosphorylase, as the hormone (see Sutherhmd and Robison, 1968). Soon afterwards, criteria were established to show whether a hormone or neurotransmitter exerts its actions by production of a second messenger within the cell. These are still widely used as an initial indication for second

messenger involvement in cell activation and are discussed below.

~-adrenergic

f ATP ~Cyclic AMP

A-kinase(active) --~ (~),~ proteins ]

."

~lns (1,4,5)P 3 (X.-adrenergic [Z~ cholinergicE~ ~Diacylglycerot C-kinase (active) - ~

proteins

J

Phorbol esters -

Fig. 4.1. Stimulus-secretion coupling. A simplified overview of the primary external stimuli and their second messengers is depicted. All second messengers activate specific protein kinases. Ca2+, may also act directly. Secretion represents both ion and protein release at the apical membrane. The main second messengers which have been identified in cells are the cyclic nucleotides (cyclic AMP and cyclic GMP), Ca 2+ and Ins(1,4,5)P 3 / diacylglycerol. We have called these second messengers in the sense that they are generated as a result of cell surface receptor occupancy and there is evidence that they, in turn, activate the cellular response. However, as will be seen below, other messengers are involved, either in the generation, or regulation of the concentration, of second messengers.

Second messenger formation Cyclic AMP and cyclic GMP are formed enzymatically from ATP or GTP by the action of the enzymes adenylate and guanylate cyclase respectively. Adenylate cyclase is exclusively located in the cell membrane, whereas there are both membrane-bound and soluble forms of guanylate cyclase (Schulz et al., 1989). Receptor proteins and adenylate cyclase are separated in the cell membrane until an agonist binds to the receptor causing it to become coupled to the cyclase by means of a guanine nucleotide binding protein (G-protein), which is also present in the cell membrane (Gilman, 1987). In contrast to adenylate cyclase, the membrane form of guanylate cyclase has a single membrane-spanning domain dividing the intracellular domain, which possesses the enzyme activity, from the extracellular receptor domain (Fig. 4.2; Garbers, 1989). Thus, binding of an agonist to its receptor causes direct activation of the enzyme without the need for coupling via a Gprotein. G-proteins are heterotrimeric, consisting of an 0~, 13 and y subunit. Several different G-proteins have been identified, predominantly by cDNA cloning (see Birnbaumer, 1990) and it is clear that there are distinct G-proteins which lead to activation of different second messenger systems. In the case of adenylate cyclase, one G-protein (Gs) couples receptors to enzyme activation and a separate one (Gi) couples receptors causing inhibition. In either case the mechanism of coupling is similar: binding of an agonist to its receptor favours association with the G-protein which drives the binding of GTP to the a subunit, in exchange for GDP (Fig 4.2). Two views as to the sequence of subsequent events have been presented in recent years (Gilman, 1987; Birnbaumer, 1990), but it is


33

agreed that the t~ subunit, with GTP bound, is the species which activates adenylate cyclase after it has dissociated from the [37 complex and from the agonist receptor complex. Hydrolysis of the bound GTP leads to reversal of activation of adenylate cyclase and it is this event which cholera toxin inhibits, thus resulting in permanent activation of adenylate cyclase.

Receptor

iAdenylyl cyclase (inactive)

G-protein~

GDp

- 200kDa, of the order of CFTR.It is noteworthy in the context of predicting the structure of CFTR that, in the original deduced structure of the Na + channel, 4 membrane spanning segments were predicted (Noda et al., 1984) whereas this was later modified to 6 on the basis of comparison with the electric eel channel and detailed knowledge of ion movement through the channel (Noda et al., 1986). Although CFTR lacks a highly-charged hydrophobic sequence, common to both the Na + and Ca 2+ channels (Noda et al., 1984; Tanabe et al., 1987) and thought to be important for voltage sensing, and contains the NBF and R domains, it is interesting that a short region within the R domain of CFTR is highly homologous to a cytoplasmic portion of the rat brain and electric eel Na+ channel.

In contrast to voltage-activated channels, the ligand-gated channels are muhimeric: for example, the functional GABA-activated CI- channel consists of 4 subunits and the acetylcholine receptor 5 subunits clustered in the membrane. In both cases the channels are heteropolymeric with the different subunits having considerable sequence homology (Stroud and Finer-Moore, 1985; Schofield et al., 1987) and molecular weights much lower (40-60kDa) than CFFR or voltage activated channels.


53

There is no evidence at present to rule out the possibility that CFTR is part of a multisubunit complex in the membrane and its putative cytoplasmic domain suggests a regulated function more analogous to a ligand-gated than a voltage-activated channel. Ligand- activated channels are depicted to possess clusters of charged residues at the ends of the membrane-spanning sequences (Schofield et al., 1987; Imoto et al., 1988). For the acetylcholine receptor (cation channel) these are negatively-charged and for the GABA el- channel, positive, leading to the suggestion that they are important for ion selectivity and regulating conductance. Indeed, site-directed mutagenesis experiments showed that, replacing negative with positively charged residues on the acetylcholine receptor reduced the conductance of the channel when expressed in Xenopus oocytes (Imoto et al., 1988). Some clustering of positively charged residues is predicted on the cytoplasmic side of the putative membrane-spanning sequences in CFTR (Riordan et al., 1989) as well as some charged residues within the transmembrane regions themselves. However, these features are not sufficient to establish whether or not CFTR functions as an ion channel. It is however worth noting that, whereas the plasma membrane GABA- and acetylcholine-activated channels have large extracellular domains, involved in ligand binding, intracellular channels such as the Ca2÷-release channel of muscle T-tubules (ryanodine receptor; Takeshima et al., 1989) and the Ins(1,4,5)P3-activated Ca2+-release channel from cerebellum (Furuichi et al., 1989) possess large cytoplasmic domains in which sites for ligand binding and phosphorylation probably reside (Fig. 6.4). The GABA-activated el- channel also possesses a putative site for cyclic AMPdependent phosphorylation (Schofield et al., 1987). The functional intracellular channels are also envisaged as consisting of multimeric complexes, as deduced from electron microscopic and density gradient centrifugation studies (Mignery et al., 1989). Much less is known about the structure of epithelial el- channels, since the gene(s) coding for them have not yet been cloned. Landry et al. (1989) have purified 4 proteins of molecular weights of 97, 64-61, 40 and 27kDa) from bovine trachea which, when reconstituted into lipid bilayers showed CIchannel activity. More recent data suggests that the 61-64kDa protein has channel activity (A1Awqati et al., 1990). However, as discussed in Chapter 3, it is not clear whether this el- channel is involved in secretion, nor whether its function is defective in CF. Clearly, the molecular weights of the component proteins do not correspond to CFTR and on balance, current predictions favour the view that CFFR is not a plasma membrane ion channel of any type currently known (Hyde et al., 1990). In addition, there is no evidence from the electrophysiology of epithelial cells that ion secretion or absorption is driven by ATP except in the indirect sense of its coupling to the Na+/K+ATPase.

Summary of Hypotheses The most common mutation in the gene (AFs08) results in alteration in the ATP binding region of CFTR, suggesting that ATP binding and/or hydrolysis is important in its function. CFFR is also likely to be regulated by phosphorylation; the AF508 mutation might either mask a phosphorylation site or inhibit involvement of ATP in autophosphorylation. The following hypotheses are emerging as to how the mutation in the CF gene leads to the abnormalities in function seen in CF epithelial cells. (1) CFTR might have a function similar to that of P-glycoprotein whereby it normally transports a substance out of the cell. In CF this transport function is defective, the substance accumulates and disrupts secretory activity. There is evidence of structural similarity of CFTR with P-glycoprotein and recent data suggests that el- channels are inhibited by a cytoplasmic factor (Kunzelmann et al., 1989); the type of molecule transported by CFTR however is unknown. The hypothesis showing removal of an inhibitor (I) of el- channel opening by normal CFTR is depicted in Fig. 6.4A.

54


STIMULATED CELL A

CF

NORMAL

CFTR ._.~

CI

i

Protein

CFTR

ADP

B

CFTR

NORMAL

CF

CI m

CFTR

Fig. 6.4. Hypotheses for the involvement of CFTR in control of epithelial CI- and protein secretion. A. On stimulation, in the normal cell, CFTR transports inhibitor (I) out of the cell (this might be dependent on ATP hydrolysis and require phosphorylation of CFTR) allowing the cell to secrete CIand proteins; in the CF cell, the mutation in CF'I~ renders it unable to export I and C1- transport and protein secretion remain inhibited. B. On stimulation, in the normal cell, phosphorylated CFTR interacts to allow CI- channel opening and protein secretion. In the CF cell, defective phosphorylation of CbTR leads to inability to transport CI- and protein.


55

(2) In view of its structure, CFTR is unlikely to be an apical membrane C1- channel; C1- channels also do not require direct ATP coupling. Cell physiological evidence (Chapter 5) suggests that it could be a protein associated with the CI- channel which regulates its activity. The finding that introduction of a normal CFTR gene into CF cells corrects the I]-adrenergic defect in regulation of C1- channel opening (Rich et al., 1990), confirms a primary link, between autonomic disturbance in CF cell secretion and a mutation in the CFTR gene. Figure 6.4B depicts the hypothesis whereby an interaction with CFTR (shown as a conformation change in the protein as a result of phosphorylation) regulates protein secretion and C1- transport. How CFTR interacts with CIchannels is not clear, however, there is also the possibility that a fragment of CFTR (see (4) below) directly interacts with the secretory process. (3) CFTR could be a plasma or intracellular membrane protein which is involved in signal transduction or translocation, packaging and secretion of mucins. (4) In view of the defective phosphorylation of a 61kDa protein in CF cells (Chapter 5), which has properties in common with the first cytoplasmic domain of CFTR and cross reacts with an antibody raised against an R-domain peptide of CFTR (Pereira, Dormer, McPherson, 1990, unpublished data), it is possible that this fragment is generated within the cell either as a result of alternative splicing of CFFR mRNA or posttranslational processing following cell activation. Different CFTR mRNA sizes are seen in epithelial tissues (Riordan et al., 1989) but may represent more subtle splicing changes. The precise role of CFTR in epithelial cell function and the consequence of a specific mutation within the CF gene will be confirmed with isolation and characterisation of the CFTR protein and studies of expression of CFTR in vivo and in vitro, as is discussed in the next chapter.

Chapter 7

CF Models

Pharmacological Before the identity of the CF gene was known, pharmacologically induced animal models were invoked by chronic injection of agents which disturb the autonomic nervous system. These were either mimetic agents such as isoproterenol or pilocarpine, or agents such as reserpine which deplete neurotransmitters. Administration of the 13-agonist isoproterenol (Mangos, 1969) led to salivary gland enlargement and the saliva was turbid, with a raised concentration of macromolecules and Na +, probably due to impaired reabsorption by the ducts. The parotid gland was shown to produce a lower rate of fluid secretion in response to pilocarpine than in untreated rats. Enlargement of submucosal glands and increased number of goblet cells in the airways was also shown to result from chronic treatment with isoproterenol (Sturgess and Reid, 1973). In terms of the composition of the glands, both isoproterenol and pilocarpine treatment increased the Ca 2+ and protein content of the submandibular gland (Wood and Martinez, 1977; Muller et al,, 1985a) but not the pancreas (Muller and Roomans, 1985). Since CF epithelial cells have defective secretion in response to 13-adrenergic stimulation (McPherson and Dormer, 1987; 1988a;b; McPherson and Goodchild, 1988), it is perhaps not surprising that desensitization produced by chronic isoproterenol treatment showed similarities to CF. However, the mechanism of desensitization by isoproterenol in vitro (Bradbury and McPherson, 1987) is different from the abnormality observed in CF cells. Although in some cell types such as the airways submucosal glands, reduction in [3-receptor numbers has been demonstrated in CF patients (Sharma and Jeffery, 1990), it is not clear that this is related to the [3adrenergic defect common to all CF epithelial cells which is beyond the site of cyclic AMP formation (McPherson and Dormer, 1988; McPherson and Goodchild, 1988). Reserpine treatment was shown to cause a number of changes in exocrine glands which resemble those seen in CF. The submandibular gland showed precipitated material in the ducts and an increased mucus content in the acinar cells (Martinez et al., 1975a; Muller et al., 1985b) which resulted in increased Ca 2+ content (Muller et al., 1985b). The intestine was also shown to have a higher mucus content (Forstner et al., 1981) and the pancreas to contain more total protein and Ca 2+ (Wood and Martinez, 1977; Perlmutter and Martinez, 1978). Both the submandibular gland and the pancreas showed a decreased flow of fluid when stimulated b~( their respective secretagogues and the secretions contained an elevated concentration of Na +, Ca z+ and total protein (Martinez et al., 1975b; Perlmutter and Martinez, 1978). In terms of specific secretory mechanisms, some intriguing parallels with the abnormalities found in CF epithelial cells have been described. Thus, parotid cells from reserpinised rats showed a decreased secretion of amylase in response to isoproterenol (Maninez et al., 1979), although the increase in cyclic AMP levels was not reduced 56


57

(Watson et al., 1984). In addition, both pancreatic and submandibular acinar cells showed decreased C1- transport responses when isolated from reserpinised rats (Martinez and Cassity, 1985b; Martinez and Martinez, 1988).

Genetic One of the consequences of the isolation and sequencing of the CF gene is the possibility of using the knowledge to develop a genetic model of the disease. However, at present, it is not known whether the major AFs08 mutation leads to an absence of CFTR or a normal amount of altered protein, nor what is the effect of the large number of other mutations detected. Although it is assumed that an altered CF protein is produced, since mRNA is present in affected CF epithelia (Riordan et al., 1989), it is possible that the mRNA is unstable in the cytoplasm or even if translated, the resulting protein may fail to fold properly and be degraded in the ER. Thus, as yet, it is not possible to predict whether complete disruption of the gene will mimic CF or whether a specific mutation such as AF508 will have to be introduced. Current technology for the specific manipulation of the genotype will involve either direct microinjection of the mutant gene into a fertilized egg which is implanted and brought to term to produce a transgenic mouse (Palmiter and Brinster, 1986) or, specifically knocking out the CF gene in pluripotent embryonic stem cells, injection of cells back into a blastula which is implanted to produce a mutant mouse (Mansour et al., 1988; Capecchi, 1989). Earlier experience with the transgenic mouse technique showed little ability to control expression of an inserted gene. Thus, Hammer et al. (1984) introduced the rat growth hormone gene into mice with hereditary dwarfism. 6 out of the 7 mice which carried at least one copy of the gene, showed levels of serum growth hormone 6-100 times higher than normal and grew to about 1.5-times normal size. In addition, this study demonstrated the unpredictability of the physiological outcome of expressing a foreign gene since the 7th mouse, which remained small, also had high serum growth hormone levels. More recent studies suggest that, as more knowledge is gained about regions of DNA outside the coding sequence of genes, but which control their expression, these types of difficulty will be lessened. Thus, Ryan et al. (1990) attached an erythroid-specific region of DNA, which is located 50kb upstream of the human [~-globin gene to both the human - and 13sglobin genes (the latter is the mutated gene coding for sickle-cell haemoglobin). Coinjecting the constructs produced transgenic mice which expressed haemoglobin (S) at similar concentrations to those of normal haemoglobin in control mice. By crossing some of these mice with a strain of 13thalassaemic mice, animals were produced which had reduced levels of normal [~-globin, were mildly anaemic and whose red blood cells sickled when exposed to reduced oxygen concentrations. The ablity to direct a transgene to the appropriate tissue will also improve the control of expression. Many genes are tissue-specific and show expression in the appropriate tissue when introduced (see Palmiter and Brinster, 1986). An example, which is particularly relevant to CF, is the demonstration that the 5'-flanking region of the rat pancreatic elastase gene provides sufficient information for tissue-specific expression (Swift et al., 1984; Hammer et al., 1987). Thus, injection of the gene, including the flanking region, produced transgenic mice which expressed elastase at a high level in the pancreas; most other tissues had undetectable levels except for the liver and spleen where the levels were at least 1000-times lower than in the pancreas (Swift et al., 1984). Furthermore, when transgenic mice were produced which carried a construct consisting of the elastase gene flanking region fused to the 5' end of the human growth hormone gene (Hammer et al., 1987), human growth hormone was expressed only in the pancreas. This type of data opens the way for a number of strategies for producing transgenic models for CF. It may be advantageous to introduce a mutant CFTR into one tissue only e.g. pancreas, to study its effects in isolation from secondary effects from abnormal function of other tissues. Other tissue specific genes, in particular those of the airways, should be investigated for selective flanking regions with a view to carrying out the same

58

M. A. McPherson and R. L. Dormer

type of experiment as for the pancreas. Alternatively, it may be that there is a sequence flanking the CF gene which directs CFFR to the epithelia affected in CF and controls both the level of expression and tissue specificity. It should be noted however, that with transgenic techniques, since CF is a recessive disease, the model produced might well be an asymptomatic heterozygote; since expression of the mutated human gene will be in conjunction with the normal endogenous mouse gene which, it is assumed, will continue to be expressed correctly. Techniques for disrupting the CF gene might be a more promising approach to producing a CF homozygote. Recent advances in the isolation and culture of pluripotent embryonic stem cells in which a specific gene can be modified and the cells injected into a mouse blastula allow the possibility of producing chimaeric mice which, when interbred, produce a strain which is homozygous for the altered genotype (see Capecchi, 1989 for a review). In mammalian cells, when a gene is introduced into a cell, the frequency with which it replaces the homologous gene in the host (homologous recombination) is of the order of 1000 times less than that for random integration into the genome (Frohman and Martin, 1989). Thus, a vital advance in this technique was the development of a method to select the very small number of cells from the stem cell population in which the homologous gene had been replaced (Mansour et al., 1988). This involved a double selection whereby a construct was made (see Fig 7.1) in which a 10-15kb sequence of the gene to be replaced contains the bacterial gene for neomycin resistance (neo) and, adjacent to this the herpes simplex virus thymidine kinase gene (HSV-thyk). When the construct is transfected into the stem cells and integrates into the genome either randomly or by homologous recombination, these cells will be neomycin resistant and so any cells which have no integrated construct are killed by growing cells in the presence of neomycin. The inclusion of HSV-thyk in the construct allows subsequent differentiation between random and homologous integration as follows: thymidine kinase converts antiviral drugs such as Gancyclovir from an inactive nucleoside analogue to a toxic molecule. If the construct had integrated by homologous recombination HSV-thyk is lost and these cells are not killed when grown in the presence of Gancyclovir. Constructs integrated randomly will retain HSV-thyk and be killed (see Fig. 7.1). This technique has recently been used to produce a strain of mice with a disrupted ]32-microglobulin gene (Zijlstra et al., 1989) and one in which the insulin-like growth factor II gene was disrupted and which showed lower growth rates than controls (DeChiara et al., 1990). It has already been shown that mice possess a gene with high homology to CFTR (Wainwright et al., 1990); by combining transgenic and mutant mouse techniques it may be possible to induce a genotype having the same mutation as in the CF gene (e.g AF508) rather than one in which there is complete absence of gene expression which might be inappropriate for CF.

Cultured Cells Development of methods for culturing human epithelial cells and the production of virally transformed immortalised cell lines (Jetton et al., 1975; Gruenert et al., 1988; Lee and Dessi, 1989) have facilitated experiments allowing incorporation of the normal or mutated CF gene (Rich et al., 1990) or protein into an appropriate cell. This will enable determination of the function of CFTR and devising site(s) of intervention to correct the secretory abnormality. Models of CF phenotype have been observed in epithelial cell lines; thus, a subculture derived from a human colonic adenocarcinoma cell line was shown to respond, with an increased short circuit current due to C1- secretion, to stimulation by ionophore A23187, but not to carbachol, lysylbradykinin, VIP or forskolin (Cuthbert et al., 1987). However, as in CF cells, forskolin gave a normal increase in cyclic AMP concentration. It is not known whether the altered function correlates with altered expression of CFFR. In other cell models, isoproterenol-stimulation of mucin secretion in submandibular acinar cells was abolished by introduction of the calcium chelator, BAPTA into the cytoplasm at a concentration which is unlikely to lower basal Ca 2+

59


concentrations, but would not allow the cell to reach a stimulated level (McPherson e t al., 1990). The most likely mechanism for such an inhibition is at a site distal to cyclic AMP formation; as is seen in CF cells. These types of cell model should lead to a greater understanding of how the secretory pathway is controlled and the role of CFTR in this process.

Target gone in ES cells ~'//A

F_L_ 1

~nsfect

l Onchanged , I

with vector

~

..........

Target gone sequences

~

Pl~v/nymlolne

__ Kinase gone

IIIlllll

~!iiiiiiii:ili:!!~:!!~ t

rNsi%r~YnCcnegone

\

\

....

Random Intergration

l !ilq

KdledbyNeomycin

KilledbyGaneylovir

I

IIIIIIii

I

Homologous Recombination Target Gone Disruption

Inject; implant

I Chimaeric mice

I

Breed with same strain as foster mother l

Homozygoue 4 for disrupted gone Interbreed

Heterozygous I for disrupted gone

I

Fig. 7.1. Technique for disruption of genes by homologousrecombination. Adapted from Mansour et at. (1988). In terms of gene transfer experiments, a normal CFTR gene has been introduced, by viral transfection, into CF airway epitelial cells (Rich e t al., 1990). This was shown to correct the defective ~-adrenergic (cyclic AMP-mediated) regulation of C1- channel opening in CF cells (see Chapter 5). Non-mammalian cells, in particular, the Xenopus oocyte are useful model systems into which foreign DNA can be directly microinjected and expressed, as previously shown for a number of genes including those coding for ion channels (Schofield e t al., 1987). This cell type may not

60


express endogenous CFTR and could therefore be appropriate for expression of normal and mutated CFTR genes to compare their function, for example whether they activate C1- channels. However, the Xenopus oocyte may not have the same regulatory mechanisms as those seen in mammalian cells. Insertion of normal CFTR protein into CF cells or altered protein into normal cells to determine its function is also a possibility using either liposomes (Wang and Huang, 1987) or hypotonic swelling (Dormer, 1983; Bradbury et al., 1989). Since it may be difficult to incorporate CFTR in the correct cellular location, an extension of this approach is to introduce antibodies against CFTR into the cell. There are many examples of antibodies directed against the active site of molecules. The demonstration that fluorescently-labelled antibodies can be incorporated into the cytoplasm of submandibular acini (Bradbury et al., 1989) affords hope that this technique will be complementary for determining the function of CFTR and sites of intervention to correct the secretory abnormality in the CF cell. It is clear that a combination of molecular genetic and cell physiological approaches will be required to elucidate the function of the CF gene protein and how a mutation in the CF gene leads to the abnormal physiology of CF epithelial cells and to the manifestations of the disease.

Chapter 8

Summary and Perspectives

Implications of CF Gene Discovery The most immediate benefit from the CF gene identification will come from the ability to carry out carrier detection and prenatal diagnosis in families which carry the most common mutation, AF508 in the CF gene (see Chapter 2). As other mutations are identified, this will increase the number of families and should eventually lead to widespread detection of previously unidentified carriers in the population. CF has been recognised as a complex disease, with wide-ranging clinical symptoms. Some individuals are much less severely affected than others, with mild lung disease and little or no requirement for pancreatic enzymes. A minority have meconium ileus and liver disease is variable in the CF population. There are also individuals with borderline sweat tests, who have other clinical features of CF, and are categorised as having the disease. Although CF is a single gene disorder, it has been assumed that the reason for the clinical variation might be because of different mutations within the CF gene. However, a common mutation (AFs08) accounts for the majority of CF cases and although trends have been noted between genotype and phenotype, it is clear that factors other than the type of mutation govern severity of manifestations of the disease. Further insights into the aetiology of CF should come from the identification of other mutations within the gene. It will also be crucial to determine how expression or activity of the CF gene protein is altered for a given mutation. Cell physiological and biochemical approaches will be used to elucidate the function of CFTR, its cellular location and whether it is subject to post-translational modification. These studies should lead to determining site(s) of intervention to correct the secretory abnormality in CF cells, and the development of a rational treatment for CF patients.

Improvements in Management of CF Even without a knowledge of the nature of the CF gene or the function of CFTR, there has been much research into how CF can be treated since its recognition in the 1930's (Andersen, 1938). In the early years, CF children often died in infancy, but over the past two decades the treatment of CF patients has improved dramatically and in the U.K., survival into adolescence or early adulthood is expected in the majority of cases (Goodchild and Dodge, 1985). The quality of life for the CF patients has also improved, mainly due to earlier diagnosis (Dodge and Ryley, 1982) and better management of disease symptoms, rather than providing a cure for the disease. The development of digestive enzyme capsules containing enteric-coated granule preparations of pancreatic enzymes, which are acid resistant, has meant that CF individuals can eat normal or even high fat diets, whereas previously they were confined to foods containing very little fat (Goodchild and Dodge, 61

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1985). The greater emphasis on physiotherapy and exercise for CF individuals has undoubtedly proved to be beneficial. Improved techniques for carrying out physiotherapy, such as the forced expiration technique, which rely on breathing control rather than aggressive percussion, are very effective and enable CF individuals to carry out their own physiotherapy. This increases independence and improves the quality of life. The development of new antibiotics for combating Pseudomonas infections such as ceftazidime which has to be given intravenously; gentamicin and carbenicillin which can be inhaled, and ciprofloxacin, which is taken orally (Goodchild and Dodge, 1985; Hodson et al., 1987) have again increased the life-span of individuals. However, development of an effective antipsuedomonal which would obviate the need for intravenous therapy is still required. Meanwhile, home administration of antibiotics enables greater flexibility for the CF individual, allowing them to maintain careers in the community. Another major advance which has so far benefited relatively few individuals and which cannot be regarded as a uniform treatment for CF has been the heart-lung transplantation programme (Geddes and Hodson, 1989). In the U.K. this has been tremendously successful, with greater than 70% of patients surviving for more than one year (Scott et al., 1988; Geddes and Hodson, 1989). In these individuals the quality of life has improved dramatically and most are well and capable of working and exercising. Replacement of the CF lung with a normal lung alleviates most of the lifethreatening illness associated with CF; the pancreatic problems still exist, however management with pancreatic enzyme capsules can be achieved. This type of approach to therapy for CF however is not ideal as patients have to undergo major surgery, complications including rejection of the donor organ can arise (Geddes and Hodson, 1989; Owen and Goodchild, 1990) and as yet the programme has not been going long enough to sufficiently evaluate results. The programme is also expensive and requires sufficient donors and selection of recipients; thus it will not be feasible for the majority of patients. However, it does emphasise that strategies aimed at treating CF patients should be targetted primarily at the lung and airways cells. Overcoming the pancreatic and intestinal problems as well would be a bonus.

Prospects for Development of New Treatments Although the improved management programmes have led to a better future prospect for CF individuals, with the new knowledge of the CF gene there is a high expectancy of finding a rational treatment for CF aimed at curing the disease rather than alleviating the symptoms. Two different approaches will be used to achieve this aim: the first is to insert a normal gene or protein into the cells of a CF individual, the second is to devise a pharmacological intervention to treat the abnormality in CF, based on knowledge of the biochemical basis of the disease. The latter seems at present to provide the most realistic hope for a rational treatment for CF patients. The two approaches are discussed below.

Gene therapy Although gene therapy, that is replacement of an abnormal gene in the DNA with a normal one (or in the case of CF insertion of a normal gene into the DNA to give an asymptomatic heterozygote) seems a logical approach to treatment of the disease and has been achieved in human cells in vitro (Rich et al., 1990), this will not be a simple therapeutic procedure. In genetic diseases in which bone marrow cells are affected, the possibility exists of removing stem cells from the bone marrow, correcting the gene defect and replacing the cells such that they repopulate the bone marrow with normal cells (Friedman, 1989). In the case of a disease like CF, gene therapy might be particularly inappropriate, since it is not likely that the stem cells for the airways epithelium are readily accessible for this type of genetic manipulation. Recent work showed that genes injected into mouse muscle in vivo incorporated into the DNA of the cells and expressed the corresponding proteins (Wolff et al., 1990). Although only a small amount of protein was made and the technique was less successful for tissues other than muscle, a future possibility of injecting a normal gene into


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the lungs or airways cells of CF patients, or even better administration of DNA in a suitable vector by aerosol must be considered. The techniques however are at a very early stage of development and it is not certain that they will ever be feasible for CF because, not only do the CF cells have to take up the normal gene and incorporate it into the DNA, but also the level of gene expression has to be regulated in order to produce the correct amount of normal protein in the CF cells. With protein replacement therapy, this again requires that the not readily accessible cells from the lungs and airways of CF patients take up the correct amount of normal protein and that the protein functions correctly in the CF cell. Liposomes containing protein or DNA have been injected into mice. They have been targetted to specific tissues by incorporating antibodies against the cell surface of the appropriate tissue (Wang and Huang, 1987). Again this procedure has not met with great success, mainly because most of the liposomes are broken down in the liver and spleen before reaching their target tissue. The possibility of administering normal protein or DNA in liposomes as an aerosol preparation to CF patients could also be considered, in the hope that the liposomes will fuse with the lung and airways cells, releasing their contents. Again these techniques are at a developmental stage and it may not be feasible to get sufficient protein into the cells to achieve normal function.

Pharmacological approaches As has been outlined in the previous Chapters, there is increasing knowledge of the cellular physiology in CF and the events regulating the secretory processes which malfunction in CF epithelial cells. This research should lead to determination of how a mutation in the CF gene protein leads to the clinical manifestations of CF, with inital experiments being aimed at finding the function of CFTR in epithelial cells. The development of a pharmacological intervention to correct the secretory abnormality in CF seems at present to provide the best hope for a treatment for the disease. The pharmacological treatments tested so far, in a recent clinical trial (Knowles et al., 1990) was aimed at correcting the ion transport abnormalities in the airways (see Chapter 5). This was based on the rationale that the increased Na + reabsorption in the airways causes the dehydration of mucus and increased viscosity of the secretions, which leads to most of the clinical manifestations of CF. Thus the drug amiloride, which inhibits Na + uptake in the airways was administered by aerosol. Although significant improvements in lung function were noted in CF patients as compared to control no dramatic differences were seen. It doesn't look as if this will be the complete answer to CF, although the trial was not carried out on presymptomatic patients and it has been argued that new therapies for CF might have to be given presymptomatically. This is also an argument for carrying out the most aggressive management policies for CF patients so that their condition will be the best possible should a new treatment be developed. Other approaches are to develop drugs which stimulate CI- channel opening. So far no specific agent is available. However, recent results have shown that ATP opens apical membrane CI- channels by acting on the apical surface of the airways cell (Boucher et al., 1990). Thus it may be possible to administer ATP in conjunction with amiloride to give a better result for the CF patient. The above approaches however, may be too simplistic for two reasons: 1. that altering ion tranport in the CF lung and airways to give a greater hydration of the mucous may not be the only reason for the increased viscosity seen in CF. 2. That adding agents which are known to open or close ion channels might not give the desired results, since evidence suggests that it is the regulation of these events which is abnormal in CF and unregulated ion channel opening might lead to unforseen consequences for the individual. It may be that altered regulation of biosynthesis and secretion of mucins is fundamental to the CF abnormality as has been suggested by experiments on mucussecreting salivary glands (McPherson et al., 1986) and airways cells (Cheng et al., 1989). A greater understanding of the regulation of protein secretion and electrolyte transport in CF cells should lead

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to devising a pharmacological means of intervention to correct the secretory abnormality, thus providing a rational treatment for CF. This has to be the hope for the future for CF individuals.

Acknowledgements Our research group is supported by Cystic Fibrosis Research Trust UK, MRC, Wellcome Trust and Welsh Scheme for the Development of Health and Social Research. We are very grateful to Dr. M.C. Goodchild and Professor J.A. Dodge for their help and encouragement and for many stimulating discussions.

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