124 Cystifibrosis - 4 Thorax 1991;46:124-130 Series editor: D JShale University Department of Pharmaology, Cambridge CB2 1QJ A W Cuthbert Reprint requests to: Professor Cuthbert Abnormalities of airway epithelial funtion and the impliations of the disovery of the ysti fibrosis gene Alan W Cuthbert Of all the funtional abnormalities present in ysti fibrosis, those affeting the lung are the most serious. In 1989 researh workers in Canada and the United States reported the struture of the gene responsible for 70% of ysti fibrosis among white people.`3 I shall explore what is known about abnormal lung funtion in ysti fibrosis at the ellular and moleular level and disuss how the reently disovered geneti information may be related to the ellular defets. Although it is too early to speak of a treatment for ysti fibrosis, the inreased understanding of underlying mehanisms suggests ways in whih treatment may be approahed in the future. By means of a painstaking ombination of hromosome walking and jumping and omplementary DNA (DNA) hybridisation over half a million base pairs on the long arm of hromosome 7 band q 31 have been sequened.`3 The putative protein oded for by the DNA for the ysti fibrosis lous onsists of 1480 amino aids. The trinuleotide odon for the 508th amino aid from the N terminus (phenylalanine) was found to be deleted in 70% of patients with ysti fibrosis. On the basis of analogy and homology a tentative struture for the protein oded for by the gene has been dedued (figure 1). The protein has been named the ysti fibrosis transmembrane regulator. This protein has two related motifs, eah onsisting of six membrane spanning regions joined by a long polypeptide hain. It is proposed that very little (5%) of the protein is exposed externally and that some 80% is in the ytosol. Two features of the ytosoli domain are important. The first is a highly harged R domain with 16 potential sites for phosphorylation and the seond two nuleotide binding folds, whih may bind or even hydrolyse ATP. The phenylalanine deletion (AF508) ours in the first nuleotide binding fold. Other proteins with similar strutures and a fair degree of homology are already known, prinipally the P glyoproteins or multiple drug resistane (MDR) proteins. These proteins are responsible for the energy dependent efflux of ytotoxi drugs from ells. Inreased expression of multiple drug resistane proteins after exposure to ytotoxi Figure 1 Hypothetial struture of the ysti fibrosis transmembrane regulator. Two sets of six membrane spanning domains are joined by a long polypeptide hain. Two nuleotide bindingfolds (NBF) bind A TP. In ysti fibrosis the missing phenylalanine is in the nuleotide bindingfold nearest to the N terminus. The R domain ontains 16 potential phosphorylation sites. Note that very little of the protein is exposed to the external (upper) surfae of the ell. drugs may be a major ause of drug resistane in aner hemotherapy.4 Transepithelial ion transport in the respiratory system What goes awry in the respiratory system in ysti fibrosis and how is this related to the presene of the ysti fibrosis transmembrane regulator protein with phenylalanine deleted at position 508? It is obvious, but worth restating, that biologial systems have no way of pumping water. Water an be made to move aross biologial barriers only by osmosis-provided, of ourse, that the barriers are water permeable. Movement of fluid therefore depends on solute transport, usually of inorgani ions. In the lungs surfae liquid elaborated in the alveoli and small airways is moved by iliary ation to more proximal regions. As fluid does not aumulate in the proximal regions even though there is a 4000 fold redution in total volume and a omparable redution in surfae area, fluid must be reabsorbed as a onsequene of ion transport.5 These same proesses are vitally important at birth for onverting fluid filled lungs to respiratory organs. How do ells lining the respiratory system, Thorax: first published as 10.1136/thx.46.2.124 on 1 February 1991. Downloaded from http://thorax.bmj.om/ on 16 June 2018 by guest. Proteted by opyright.
Abnormalities of airway epithelialfuntion in ystifibrosis Figure 2 Models of eletrogeni sodium absorption (a) and eletrogeni hloride seretion (b). Cells are joined at the apial surfae by tight juntions. In (a) sodium ions enter the ell through apial sodium hannels and are pumped out of the basolateral surfae by the sodium pump (P). Potassium ions yle via the sodium pump and basolateral K hannels. In (b) hloride ions enter the ell by a basolaterally loated arrier (C) along with potassium and sodium ions. The latter two are dealt with by the pump and potassium hannels as before. Chloride ions leave through the apial surfae via hloride hannels sensitive to yli AMP. Counterions, hloride in (a) and sodium in (b), move through paraellular pathways. A-amiloride. partiularly in the more proximal regions, effet fluid absorption? Two types of transporting mehanism are depited in figure 2- namely, eletrogeni sodium absorption and eletrogeni hloride seretion. Both proesses are powered by the sodium pump, a sodium-potassium ativated, magnesium dependent ATPase. The transporting epithelial ells are asymmetrial-that is, the apial and basolateral membrane domains differ in their properties in suh a way that the moleular apparatus of transport, ion hannels, ion pumps, and exhange mehanisms are exlusively assigned to one membrane domain or the other. These moleular entities are prevented from passing from one domain to the other by the tight juntions between individual ells. How an individual ell is able to insert these protein moleules into the orret membrane domain remains an intriguing and largely unsolved problem. Sodium absorption is dependent on the presene of seletive sodium hannels in the apial membrane (fig 2a), whih an be speifially bloked by drugs suh as amiloride and triamterene.6 Although sodium hannels are present there will be net sodium influx into the ell only if a favourable eletrohemial gradient exists. Sodium ions are expelled from the ell aross the basolateral membrane via the sodium pump, whih has the stoihiometry of 3+ to 2K+ ions. The transfer of the sodium ation aross the epithelial barrier reates a favourable eletrial gradient for the movement of the hloride ounteranion, whih moves passively aross the tissue, probably by a paraellular route. Potassium ions establish eletrohemial equilibrium aross the basolateral fae via potassium hannels, whih in many systems are sensitive to the onentration of intraellular alium. Eletrogeni sodium absorption an be prevented by inhibitors of the sodium pump, suh as ouabain and other ardia glyosides. There appear to be few endogenous substanes that an regulate this sodium absorptive proess in respiratory epithelium, though 125 adrenaline is known to stimulate the proess in the perinatal period.7 Normally eletrogeni hloride seretion is not present in respiratory epithelium, but it an be ativated by beta agonists and other agents that inrease intraellular yli AMP, prostaglandins, and bradykinin. The first step in hloride seretion requires that hloride is aumulated in the ell to a level greater than expeted from the eltrohemial gradient. The ell does this by using a sodium-potassium hloride otransporter with the stoihiometry +: K+: Cl- 1:1:2 (fig 2b). The energy stored in the eletrohemial gradient for sodium between the outside of the ell and the interior provides the energy soure for driving potassium and hloride uphill into the ell. The sodium and potassium ions that enter the ell along with hloride are dealt with by the sodium pump and by equilibration through basolateral K+ hannels respetively. Chloride is then free to move out of the ell, aross the apial membrane, and down its eletrohemial gradient, provided that the hloride hannels are open. Agents that inrease yli AMP inrease the open state probability of these hannels. Thus the proesses of hloride seretion lead to a transfer of harge (anions) aross the tissue suh that a favourable gradient for aompanying ounteration movement is generated. The main ounterions will be sodium ions moving mainly through paraellular routes. Both the absorptive and seretory proesses that have been desribed will influene water movement, inwards and outwards respetively, aross the epithelium. This is possible beause respiratory epithelium is water permeable. This is not so for epithelia with low water permeability (for example, sweat dut), where salts an be absorbed without onsequent fluid movement. The presene in the pulmonary system of these transport proesses, with a predominant, unregulated absorptive proess plus a seretory proess that an be ativated and regulated by autaoids and transmitters, provides the opportunity for fine tuning of the thikness of the fluid (sol) phase lining the proximal airways. The ontrol of the thikness of the fluid lining has not been definitely demonstrated, but it is known that both these proesses are present in epithelium lining the proximal airways and, more importantly, how they are modified in ysti fibrosis. Abnormalities of epithelial ion transport in ysti fibrosis The first insightful observation in relation to the transport funtion in ysti fibrosis airway epithelium was that the potential differene aross the nasal muosa and bronhi of patients with ysti fibrosis was around twie that of subjets not suffering from ysti fibrosis.8 On its own this result is diffiult to interpret beause either inreased sodium absorption or redued hloride ondutane at the apial surfae would give this result. When amiloride is superfused into the apial surfae of nasal epithelia in vivo the potential Thorax: first published as 10.1136/thx.46.2.124 on 1 February 1991. Downloaded from http://thorax.bmj.om/ on 16 June 2018 by guest. Proteted by opyright.
126 AMP] Cl Normal Cl CF Cl [CAMP Cl_l Figure 3 Summary of the differenes between airway epithelium in normal and ysti fibrosis tissues. In ysti fibrosis sodium absorption () is exaggerated and hloride seretion (Cl) is bloked, even in the presene of seretagogues that inrease yli AMP (AMP). In normal epithelium seretagogues inrease hloride seretion whereas in ystifibrosis sodium absorption is enhaned. Exessive dehydration may remove the sol phase, depositing muus flakes with adherent bateria on to the ilia. differene falls. Both the absolute fall and the perentage inhibition of the nasal potential differene was greater in patients with ysti fibrosis than in normal ontrols9 and this effet ould be mimiked by superfusing sodium free solutions. In ontrast, superfusion with hloride free solutions inreased the luminal negativity, but onsiderably more in normal subjets than in patients with ysti fibrosis.9 These additional findings support the notion that both inreased sodium transport and redued apial hloride ondutane are responsible for the potential differene in the airway epithelium in ysti fibrosis. Monolayers of ultured nasal epithelial ells from ontrols and patients with ysti fibrosis showed similar potential hanges when subjeted to amiloride or to low sodium or low hloride onditions.'0 This means that ells retain their ysti fibrosis harateristis through many generations of ell division in ulture, exatly what is expeted if abnormal harateristis are tied to a geneti defet. Sine these early, largely in vivo experiments, various lines of evidene have onfirmed that in airway epithelium from patients with ysti fibrosis sodium absorption is enhaned and apial hloride permeability is low or absent, the latter preventing hloride seretion in response to seretagogues that raise intraellular AMP. The detailed evidene for this annot be given here, but the entral findings are summarised below with key referenes. 1 Isolated epithelial sheets from reseted nasal turbinate material or nasal polyps were used to measure ative sodium absorption. The rate of sodium transport in ysti fibrosis tissue was about twie that in ontrol tissue.'""'3 2 In ultured airway epithelial ells apial hloride permeability was lower in ysti Cuthbert fibrosis ells than in non-ysti fibrosis ells.'4 '5 3 By appliation of various agents that ativate adenylate ylase (beta agonists, forskolin) to monolayer ultures of airway epithelial ells hloride seretion was stimulated in normal but not ysti fibrosis ells. In ysti fibrosis monolayers beta agonists further enhaned sodium absorptive proesses. These differenes ould not be explained by differenes in the generation of AMP or in the amount of AMP dependent protein kinases in the tissue. '"'" 4 Bradykinin and A23 187, both of whih inrease intraellular alium onentration (Ca-), are able to eliit transient hloride seretory responses in ysti fibrosis epithelia.'9 It is, however, possible that raised Cai ativates basolateral K+ hannels, leading to hyperpolarisation of the apial membrane, thus inreasing the eletrohemial potential for efflux, even though the membrane has a low hloride ondutane. Figure 3 provides a summary of the omparisons in transporting properties between ysti fibrosis and normal respiratory epithelium, plus the effets of different types of seretagogues. Possible onsequenes of transporting abnormalities in ysti fibrosis Muoiliary learane depends on the funtioning of ilia within the sol phase lining the respiratory passages. Not only is fluid moved toward the proximal airways but muus and entrapped foreign partiles are moved to a position from whih they an be eliminated from the lung. Exessive solute transport out of the airways, with onsequent fluid absorption, may redued the sol phase, depositing thik, visid muus on the ilia. Muus deposits are diffiult to dislodge and provide a breeding ground for Staphyloous aureus and Pseudomonas aeruginosa. Although this is a reasonable hypothesis there is little definitive evidene to support it. The reason is a tehnial one-it is diffiult to sample fluid from the airways without evaporative losses, making aurate determination of omposition very diffiult. The hemial omposition of sereted muus is known to be altered in ysti fibrosis, with inreased sulphation of high moleular weight glyoonjugates.20 As ysti fibrosis is a single gene defet it beomes inreasingly diffiult to explain how properties as diverse as muus omposition and hloride ondutane are related to a single abnormal form of the ysti fibrosis transmembrane regulator, though abnormal hloride transport may inrease the onentration of intraellular sulphate and inrease substrate driven sulphation of glyoproteins. Moleular orrelates of abnormal transporting properties Path lamping is a relatively new tehnique that allows the funtional ativity of single ion hannels to be examined. Theoretially the onept is straightforward. First devise a way Thorax: first published as 10.1136/thx.46.2.124 on 1 February 1991. Downloaded from http://thorax.bmj.om/ on 16 June 2018 by guest. Proteted by opyright.
Abnormalities of airway epithelialfuntion in ystifibrosis 127 of isolating a single ion hannel so that its ativity an be monitored, without interferene from the thousands of other ion hannels present in a ell membrane. Seondly, find a way of deteting whether the hannel is open or losed. This is done by monitoring the flow of ions through the hannel with sensitive urrent amplifiers. Ion hannels allow millions of ions to pass eah seond, giving rise to urrents in the pioampere (10-12 A) range. By measuring the relation between the urrent and the voltage aross the hannel and taking aount of the ion onentration either side of the membrane you an assess hannel seletivity. An all important fator is the reversal potential (the potential at whih the urrent through the hannel reverses its diretion); appliation of the Goldman- Hodgkin-Katz equation and omparision of atual and theoretial values allow the hannel to be designated as seletive for hloride, sodium, potassium, or alium. To isolate a single ion hannel a fire polished glass eletrode with a tip diameter of a few mirons is gently pushed against the surfae of a ell. A self sealing proess ours suh that the resistane between the onduting fluid in the eletrode and the bathing fluid outside is very large (gigaohm seal). The path so isolated will ontain a few ion hannels, or preferably a single one. Reordings are made of the urrents flowing aross the path either in the ell attahed mode or by pulling the path off the ell. Figure 4 shows a path lamp reord from a hloride ion hannel from a human ysti fibrosis sweat gland ell. Diagrammati representation of major findings in ysti fibrosis and airway epithelial ells is given in figure 5, whih shows idealised diagrams of path lamp reordings. Figures 5a and 5b shows the usual features seen with path lamping. The urrent flowing through a partiular type of ion hannel is onstant at a given voltage-that is, the hannel ondutane is harateristi of the hannel, and hannels are either open or losed. If two hannels are present in a path and are open simultaneously then the urrent flowing is twie that of a single hannel. By ontrast, the duration of the open and losed periods varies even for a single hannel. In the simplest systems analysis of path lamp reords shows that open and losed time histograms are fitted by single exponentials, suggesting that a single proess is responsible for transitions from open to losed. Two groups have reported their findings on hloride hannels in airway epithelial ells using the path lamp tehnique.2 22 In the ell attahed onfiguration hloride hannels were rarely seen unless the ell was ativated by agents that inreased AMP. After stimulation there was an inreased probability of hloride hannel opening in normal ells (fig 5) but not in ysti fibrosis ells (fig 5d). Again with the ell attahed onfiguration, the alium ionophore A23187 was able to ativate hloride hannels in both normal and ysti fibrosis ells. Isolated pathes from ells ativated by AMP generating seretagogues showed hloride hannel ativity (fig 5ei), whereas pathes from similarly I 4 pa 10 ms Figure 4 Path lamp reordingfrom an apial membrane path showing the ativity of a single hloride seletive hannelfrom a ysti fibrosis sweat gland ell. Channel opening is in the upward diretion, indiating hloride ion flowfrom outside the apial surfae to inside. The solution bathing both sides of the path ontained potassium (140 mmol/l), sodium (7 mmol/l). The holding potential was 70 mv with the outside apial surfae negative. treated ysti fibrosis ells were eletrially silent (fig 5eii). Chloride hannels ould be ativated in ysti fibrosis pathes either by strong depolarisation or by exposure of the ytosoli fae to alium ions (fig 5eiii). The effets of these last two manoeuvres are not (a) (b) () (d) (ei) (eii) (eiii) (fi) (fii) L Figure S Theoretial path lamp reordsfrom hloride hannels. indiates the urrent level at whih the hannel is losed. The path may ontain one (a) or more than one (b) hannel. Ativation of a respiratory epithelial ell with yli AMP (AMP) inreases the probability that the hloride hannels are open. In normal ells hannel ativation an be seen in the ell attahed onfiguration () but not with ystifibrosis ells (d). When the path is pulledfrom ativated ells hannel ativity ontinues in pathesfrom normal ells (ei) but is still absent in pathesfrom ysti fibrosis ells (eii). Pathes from ystifibrosis ells an, however, be ativated by strong depolarisation (eiii). When a path is held at a potential that prevents hloride hannel ativation opening an be indued by a oktail ofa TP, AMP, and the atalyti subunit ofprotein kinase A (added at 0) in pathesfrom normal (fi) but not ysti fibrosis ells (fii). Thorax: first published as 10.1136/thx.46.2.124 on 1 February 1991. Downloaded from http://thorax.bmj.om/ on 16 June 2018 by guest. Proteted by opyright.
128 Cuthbert immediate or neessarily reversible and it is not lear how ativation is ahieved. Kunzelmann et al2' have shown that if isolated pathes are formed at 37 C rather than at room temperature there is immediate ativation of hloride hannels in ysti fibrosis ells without the need to apply strong depolarisation. He has argued that regulation of the hloride hannel is defetive rather than the hannel itself. Possibly the ytosol in ysti fibrosis ontains a toni inhibitor of the hloride hannel that is removed one an inside out path is formed. Finally, as shown in figure 5f, when apial membrane pathes are held at room temperature and at a voltage inhibiting hannel ativation, addition of ATP and the atalyti subunit of protein kinase A auses hannel ativation in normal but not ysti fibrosis membranes. Thus there appears to be a phosphorylation defet at a stage later than either AMP generation or the ativation of protein kinase A.24"2 There are two other findings with a bearing on this problem. Firstly, the path lamp findings have now been onfirmed in an immortalised, virally transformed epithelial line developed from ysti fibrosis airway ells. This establishes that the distintive ysti fibrosis phenotype is preserved in ells after many generations of ulture in vitro,26 and therefore annot be related to the infeted onditions in ysti fibrosis airways. Seondly, apial membrane hloride hannels inorporated into lipid bilayers show properties similar to those in normal membranes, but undergo a rapid run down. Ativity an be restored by addition of the phosphorylating "oktail" inluding the atalyti subunit of protein kinase A, indiating that a phosphorylation event is onerned in the funtioning of airway hloride hannels.27 Protein kinase C an also ativate hloride hannels in airway epithelial ells, but again regulation by this kinase is defetive in ysti fibrosis.28 Whatever method has been used to ativate ysti fibrosis hloride hannels in vitro their properties have proved to be idential to those of normal hannels-that is, ondutane of around 50 ps at 0 mv, an outwardly retifying urrent-voltage relationship, and a Cl- to + seletivity ratio of 10:1. Far less is known about sodium hannel mehanisms in airway ells. The mean hannel lifetime for sodium hannels in ysti fibrosis ells is twie that ofnormal hannels, providing a orrelate for the inreased sodium reabsorption in ysti fibrosis.29 It has also been shown that the marosopi Kd for amiloride is inreased and there is an altered relation between sodium onentration and sodium transport in ysti fibrosis epithelium.'0 These latter studies were made with sweat dut epithelium, however, not airway epithelium. Nevertheless, a piture is emerging of altered moleular harateristis affeting both hloride and sodium hannels in ysti fibrosis. From the path lamp data it is diffiult to argue that the sodium hannel effets are a result of aberrant hloride transport, yet both alterations in hannel properties are a onsequene of a single gene defet. Future therapeuti strategies An experiment that many must now be attempting is to transfet various ells with the DNA for ysti fibrosis transmembrane regulator. This approah may show whether or not the ysti fibrosis transmembrane regulator is the hloride hannel. This seems a little unlikely as altered sodium hannel harateristis are retained in isolated pathes. More probably it will be shown that transfetion of ysti fibrosis ells with the ysti fibrosis transmembrane regulator DNA will orret the ion hannel abnormalities. In the long term transfetion of airway epithelium in patients with DNA in a retrovirus vetor may be possible. Insufflation of the appropriate material into the respiratory trat may be easily arranged and onsiderable improvement might be envisaged even from transfetion of only a fration of the affeted ells. Major obstales need to be overome before ethial permission is likely to be given, not the least of whih is the possibility of swithing on ellular onogenes. An alternative approah is to orret the transport irregularities with agents ative from the apial surfae. Nebulisation provides a route of administration that is likely to onfine the pharmaologial effet to the airways. Amiloride, an epithelial sodium hannel bloker, is an obvious andidate and has been tried.3' Fourteen volunteer adults with ysti fibrosis took part in a study in whih amiloride solution or ontrol vehile was administered by nebuliser in a double blind rossover study with two 25 week treatment periods. The mean (SEM) loss in fored vital apaity was redued signifiantly from 3-4 (1 1) ml/day with vehile to 1-4 (07) ml/day with amiloride. Furthermore, the rheologial properties of the seretions returned to virtually normal values during amiloride administration, though baterial ontamination was unmodified. This trial raises the possibility that prophylati treatment for young patients with ysti fibrosis before aute respiratory symptoms have developed may be worthwhile. Analogues with greater poteny or a longer duration-for example, benzamil32- might be more useful. As we have seen, evidene suggests that, at least in airway epithelia, hloride seretion fails in response to seretagogues working via AMP whereas agents that inrease Cai are still effetive. Simultaneous inhibition of sodium absorption and stimulation of hloride seretion may prove even more effetive than the former alone. There is an added inentive to develop this strategy beause when amiloride bloks the apial sodium hannels in airway epithelia a profound hyperpolarisation of the apial membrane results, exatly what is required to promote hloride exit from the ell provided that apial hloride hannels an open. Thus a small inrease in hloride ondutane may have an amplified effet on hloride seretion in the presene of amiloride. Several ways of inreasing hloride ondutane may be envisaged, via novel agents that raise Ca, or development of hloride hannel openers working independently of normal regulatory mehanisms. Agents of the former Thorax: first published as 10.1136/thx.46.2.124 on 1 February 1991. Downloaded from http://thorax.bmj.om/ on 16 June 2018 by guest. Proteted by opyright.
Abnormalities of airway epithelialfuntion in ystifibrosis type are known (for example, thapsigargin33) but are only experimental tools. Bradykinin promotes hloride seretion in many types of epithelia' and does not require AMP for its ation. Bradykinin reeptors are usually loated basolaterally35 but in airways they are loated apially.6 Bradykinin, however, also auses bronhoonst-rition, learly undesirable in ysti fibrosis, but is rapidly metabolised by tissue peptidases. Some thought should be given to developing analogues with appropriate pharmaodynami and pharmaokineti properties that may promote hloride seretion in airway epithelium. In view of the similarity between the ysti fibrosis transmembrane regulator and multiple drug resistane proteins mentioned earlier we may ask whether the ysti fibrosis transmembrane regulator performs a similar funtion, expelling unwanted endogenous substanes from ells that otherwise alter the funtioning of other systems, suh as the behaviour of sodium and hloride hannels. The number of endogenous materials whih, in exess, might have this effet is obviously large but the possibility that a toni inhibitor of the hloride hannel exists is supported by the immediate ativation of these hannels when the pathes are removed from ells at 37 C.23 Moreover, the volume of distribution of many drugs is inreased in ysti fibrosis, indiating that they an penetrate spaes from whih they are normally exluded. Is this too a manifestation of an altered funtion of the ysti fibrosis transmembrane regulator? New information about the ysti fibrosis transmembrane regulator may promote questions where there is already a body of linially relevant information ontaining important lues. For example, the immunosuppressive drug ylosporin has been shown to label multiple drug resistane proteins37 and possibly the ysti fibrosis transmembrane regulator is also labelled; if it is, is its funtion altered? This ould be studied in patients reeiving transplants and ylosporin to see whether they show any membrane harateristis of ysti fibrosis and whether these disappear as ylosporin dosage is redued. Multiple drug resistane proteins are inreased in ells, partiularly epithelial ells, when they are exposed to natural ytotoxi drugs, suh as vinblastine and vinristine. If the ysti fibrosis transmembrane regulator an also be indued, a searh for non-toxi induers may lead to a useful treatment as ysti fibrosis may be a missense mutuation, where ativity of the abnormal ysti fibrosis transmembrane regulator is not destroyed but impaired. The ysti fibrosis transmembrane regulator with phenylalanine deleted at position 508 aounts for only 70% of mutations. Other haplotypes, aounting for the other 30% of mutations, are likely to be disovered soon. This information will provide further lues about whih strutural features of the ysti fibrosis transmembrane regulator are essential for funtion. Summary Details of ion transporting abnormalities in 129 ysti fibrosis airway epithelium are now known. The entral hypothesis, that exessive drying of the airway surfaes is a primary event that leads to all the manifestations of the respiratory insuffiieny in ysti fibrosis, is not proved. The hypothesis is, however, onsistent with the known transporting abnormalities and is strengthened by the modest linial improvement produed by a strategy designed to orret the transporting abnormalities. The disovery of the ysti fibrosis gene, together with the presumed struture of the protein produt, provides a foal point that must eventually onnet the funtional abnormalities with the geneti defet. The ellular funtion of the ysti fibrosis transmembrane regulator must now be the major target in researh on ysti fibrosis. Strategies for treatment based on known ellular and moleular abnormalities are beginning to emerge but will be undoubtedly more foused one the responsibility of the ysti fibrosis transmembrane regulator is known. Addendum Sine this review was ompleted the predition made under "Future therapeuti strategies" has been realised. Complementation studies have shown that the introdution of plasmids ontaining ysti fibrosis transmembrane regulator DNA into ultured ysti fibrosis ells does indeed restore a hloride ondutane response to AMP.3839 Additionally, it now appears that the ysti fibrosis transmembrane regulator fails to be glyosylated in ysti fibrosis ells, being retained in the golgi and not transferred to the ell membrane.40 A plasma membrane loation of the ysti fibrosis transmembrane regulator is onsistent either with an MDR like funtion or with its being a diret regulator of ion hannel ativity. Results obtained in the author's laboratory were from work supported by grants from the Cysti Fibrosis Researh Trust. 1 Rommens JM, Iannuzzi MC, Kerem B, et al. Identifiation of the ysti fibrosis gene: hromosomal walking and jumping. Siene 1989;243:1059-65. 2 Riordan JR, Rommens JM, Kerem B, Zielenski J, Lok S, Plavsi N, Chou JL, Drumm ML, Iannuzzi MC, et al. Identifiation of the ysti fibrosis gene: loning and haraterisation of the omplementary DNA. Siene 1989;245: 1066-72. 3 Kerem B, Rommens JM, Buhanan JA, et al. Identifiation of the ysti fibrosis gene: geneti analysis. Siene 1989;245:1073-80. 4 Bradley G, Juranko PF, Ling V. Mehanism of multidrug resistane. Biohim Biophys Ata 1988;948:87-128. 5 Kilburn KH. A hypothesis for pulmonary learane and its impliations. Am Rev Respir Dis 1968;98:449-63. 6 Cuthbert AW, Fanelli GM. Effets ofsome pyrazine arboxamides on sodium transport in frog skin. Br J Pharmaol 1978;63: 139-49. 7 Olver RE, Ramsden CA, Strang LB, Walters DV. The role of amiloride-blokable sodium transport in adrenaline indued lung liquid absorption in the foetal lamb. J Physiol 1986;376:321-40. 8 Knowles M, Gatzy J, Bouher R. Inreased bioeletri potential differene aross respiratory epithelia in ysti fibrosis. N Engi I Med 1981 ;305: 1489-95. 9 Knowles M, Gatzy J, Bouher R. 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130 Cuthbert fibrosis regulatory epithelium. Siene 1983;221:1067-70. 12 Knowles MR, Stutts MJ, Yankaskas JR., Gatzy JT, Bouher RC. Abnormal respiratory epithelial ion transport in ysti fibrosis. Clin Chest Med 1986;7:285-97. 13 Cotton CU, Stutts MJ, Knowles MR, Gatzy JT, Bouher RC. Abnormal apial ell membrane in ysti fibrosis respiratory epithelium. An in vitro eletrophysiologial analysis. J Clin Invest 1987;79:80-5. 14 Widdiombe JH, Welsh MJ, Finkbeiner WE. Cysti fibrosis dereases the apial membrane hloride permeability of monolayers ultured from ells of traheal epithelium. Pro tl Aad Si 1985;82:6167-71. 15 Stutts MJ, Cotton CU, Yankaskas JR, et al. Chloride uptake into ultured airway epithelial ells from ysti fibrosis patients and normal individuals. Pro t! Aad Si 1985;82:6677-81. 16 Bouher RC, Stutts MJ, Knowles MR, Cantley L, Gatzy JT. + transport in ysti fibrosis respiratory epithelia. Abnormal basal rate and response to adenylate ylase ativation. 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