Physiological Basis of Cystic Fibrosis: A Historical ...€¦ · PHYSIOLOGICAL REVIEWS Vol. 79, Suppl., No. 1, January 1999 Printed in U.S.A. Physiological Basis of Cystic Fibrosis:

PHYSIOLOGICAL REVIEWS

Vol. 79, Suppl., No. 1, January 1999Printed in U.S.A.

Physiological Basis of Cystic Fibrosis: A Historical Perspective

PAUL M. QUINTON

Department of Pediatrics, University of California, San Diego, La Jolla; and Division of Biomedical Sciences,

University of California, Riverside, California

I. Introduction S3II. From Witches to Science S4

III. Research in the ‘‘Wild West’’ S5IV. The Mucus Story S5

A. Pancreas S6B. Airways S6C. Gastrointestinal tract S7D. Reproductive tract S7E. Salivary glands S7F. The cell S8

V. The Electrolyte Story S9A. Sweat gland S9B. Pancreas S11C. Airways S12D. Intestine S13E. The gene and cell S13

VI. Which Story to Believe? S14

Quinton, Paul. M. Physiological Basis of Cystic Fibrosis: A Historical Perspective. Physiol. Rev. 79, Suppl.: S3–S22, 1999.—Cystic fibrosis made a relatively late entry into medical physiology, although references to conditionsprobably reflecting the disease can be traced back well into the Middle Ages. This review begins with the originsof recognition of the symptoms of this genetic disease and proceeds to briefly review the early period of basicresearch into its cause. It then presents the two apparently distinct faces of cystic fibrosis: 1) as that of a mucusabnormality and 2) as that of defects in electrolyte transport. It considers principal findings of the organ and cellpathophysiology as well as some of the apparent conflicts and enigmas still current in understanding the diseaseprocess. It is written from the perspective of the author, whose career spans back to much of the initial endeavorsto explain this fatal mutation.

I. INTRODUCTION zens in the struggle for its control (28). It is little wonderin view of this wide range of effects and of the remarkable

During my career, cystic fibrosis (CF) has grown vig- progress made in our time in understanding the diseaseorously in science, medicine, and society. Scientifically, that it is now considered by some to be a paradigm forCF has advanced from innumerable speculations about integrating scientific, medical, and social efforts in theits cause to a precise definition of causative mutations control of a disease. But still we have no cure and noaccompanied by accurate, quantitative descriptions of effective control of its relentless destruction of the lungstheir physiological effects. Medically, the life expectancy and pancreas.of CF patients has increased from death in early childhood This review is constrained to the events and observa-to an age over 30 years (98), with a great reduction in tions that, from my viewpoint, seem to bear directly onpediatric morbidity.1 Socially, it has almost become a the development of fundamentals in our understanding ofhousehold word, enlisting the support of millions of citi- the disturbances in the physiological systems and pro-

cesses that result in the disease we call CF. I can in no1 In the United States, the gene frequency is almost 4%, and CF way deal with all of the observations, insights, and efforts

afflicts Ç1 in 3,200 live births (1 in 2,400 in the United Kingdom). Overthat have created our present understanding. An excellent20,000 patients are under the care of 113 certified CF centers alone (76,

93, 111, 248). recent review (266) covers a number of contemporary and

S30031-9333/99 $15.00 Copyright q 1999 the American Physiological Society

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PAUL M. QUINTON Volume 79S4

more recent subjects that relate fundamental abnormali- still nameless, the present day terminology for CF beganto evolve during the 1930s. These descriptions focusedties to clinical progress and gene therapy.

Over the past three decades, I have struggled, and on the pancreatic involvement and steatorrhea and oftenrelated it to a form of celiac disease, although the concur-still struggle, to understand how CF can be seen both as

a disease simply caused by abnormally thick mucus and rence of bronchopulmonary complications were noted aswell (4, 26, 95, 119). Fanconi et al. (95), in 1936, wereas a disease of clearly fundamental defects in electrolyte

transport. This struggle admittedly shapes this review. In probably first to refer to the disease as ‘‘cystic fibro-matosis with bronchiectasis’’ and recognized it as sepa-so much as this approach may be controversial because

of the inevitable biases that I carry, I hope that it might rate from celiac disease. Andersen in 1938 (4) used theterm ‘‘cystic fibrosis of the pancreas.’’ Histochemical ex-also provoke better resolution of the problem. Also, I have

taken the more personal, but less scientifically formal, aminations of other organs revealed inspissations andconcretions in the ducts and lumens of intestine and lung.approach of citing the names in most instances of the

investigators of whom I have been privileged to know. By 1945 (before the abnormality in sweat was known),Farber (96) had introduced the term mucoviscidosis toThere are others that I have not named directly; I hope

that they will be forgiving. I am indebted to them all. challenge the fledgling nomenclature that Andersen hadfocused on the pancreas. He firmly believed that thecause of the disease was a generalized ‘‘state of thickened

II. FROM WITCHES TO SCIENCE mucus.’’ This name is still widely used and often preferredoutside the English speaking world. In retrospect, bothnames were misnomers reflecting the limited view of theHistorical searches lead us to believe that at least

some of the characteristic symptoms of the disease may pathology at the time. In 1946, Andersen and Hodges (5)refined the observations and presented the first compel-have been recognized and been associated with its mor-

tality in the primitive societies of eastern Europe even ling evidence that the disease is genetic and results froman autosomal recessive mutation. Two years later, an im-before the Middle Ages. Most of our information on the

earliest indications of CF come from the persistent dig- pressive heat wave in New York resulted in a high inci-dence of heat prostration among a number of her patientsgings of Busch (40–46). A few references uncovered in

medieval folklore predict death for an infant that tastes at Columbia Hospital (136). Fascinated by this event, diSant’Agnese, also at Columbia, sought the source of the‘‘salty’’ when kissed. Such infants were thought to be

‘‘hexed’’ (46). In 1606, Alonso y de los Ruyzes de Fonteca, salt loss that had precipitated the heat prostration (73).His conclusion brought the empirical insights of primitiveprofessor of medicine at Henares in Spain, wrote that it

was known that the fingers taste salty after rubbing the folklore on salty tasting kisses into the medical literatureof the 20th century. di Sant’Agnese presented his findingsforehead of the bewitched child (3). The modern diagnos-

tic criterion of salty sweat seems to have been seen even to the American Pediatrics Society in 1953 as the lastpaper of the day which he noted ‘‘almost no one attended’’then as a premonition of sickness, emaciation, and early

death. Possibly the earliest accurate medical description (personal communication). General acceptance of the ab-normality as a fundamental component of the diseaseof the pancreatic lesion in a case of CF was given in an

autopsy report on a supposedly ‘‘bewitched’’ 11-yr-old girl took several years. But within 5 yr, it was established thatthe uniqueness of this particular abnormal physiologicalin 1595 by Pieter Pauw, professor of botany and anatomy

in Leiden, The Netherlands. He described the child as function afforded a method for accurate, accessible diag-nosis. The sweat test quickly became and remains thebeing meager with a swollen, hardened, gleaming white

pancreas (46). Anecdotal records from the mid-1600s re- most common and reliable single parameter, possibly ex-cepting recently available genetic analysis, for differentialport conditions that must have reflected steatorrhea, pre-

sumably arising from pancreatic insufficiency, another diagnosis of CF (158).However, these apparently unrelated findings set thediagnostic hallmark of the disease (40, 42, 44, 124). A few

records from the 19th century and early 20th century stage for confusion. On the one hand, it was obvious tothe pathologist that the disease was caused by abnormalassociated steatorrhea, meconium complications, and

pancreatic lesions (103, 213). Landsteiner, who also de- mucus or proteinaceous inspissations in the lumens ofpancreas, lung, and intestine. Adjectives like ‘‘thick,’’fined blood groups, published possibly the first modern

description of meconium ileus associated with a defec- ‘‘sticky,’’ ‘‘tenacious,’’ and ‘‘inspissated’’ were used liber-ally in early reports (4, 26, 95, 96, 277) and continue totive CF pancreas (156). Meconium ileus is now consid-

ered to be almost pathognomonic of the disease (216). be used (93, 94, 150, 184, 207, 255) to describe the micro-scopic changes observed in affected tissues. On the otherThe turn of the 20th century brought the first obser-

vations that began to associate lung disease with diarrhea hand, the sweat gland with its abnormally salty secretion,which produced no mentionable amount of mucus andand abnormal pancreatic function as well as reports of

congenital familial steatorrhea (103, 272). Although it was showed no structural defects, became the clearest crite-


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January 1999 CYSTIC FIBROSIS: A HISTORICAL PERSPECTIVE S5

rion for diagnosis. Any unifying hypothesis needed to ex- cellular Ca2/ in mononucleocytes was abnormal. De-creased protease activity (203, 261), arginine esteraseplain both.(202), g-glutamyl transpeptidase (19), galactosyl trans-ferase (204), a-D-glucosidase (2) activities, and a2-macro-

III. RESEARCH IN THE ‘‘WILD WEST’’ globulin (234) were all reported to be altered as well.Attention turned again to cultured cells when Breslow

and co-workers (35, 37) reported that CF fibroblasts wereIn the ensuing 20 years, the search for a basic defectthat would unify the disparate symptoms of the disease much more resistant to dexamethasone in culture than

their normal counterparts. It was reported that Tamm-followed numerous paths mainly based primarily on em-pirical observations, often completely unrelated to any Horsfall glycoprotein induced a multifold increase in alka-

line phosphatase activity in CF fibroblasts (49), that acidknown pathology. Much of the literature from the late1960s and 1970s came from what might be viewed as a phosphatase and a-mannosidase enzymes from CF cells

were more heat sensitive than normal (50), that intracellu-‘‘Wild West’’ era of research. Few things made sense, andthere seemed to be almost no guidelines. I well remember lar Ca2/ was increased in CF fibroblasts (8, 51), and that

calmodulin regulation of Ca2/-ATPase was abnormal (132).Efraim Racker’s remark in 1985 as he began an overviewof CF as an address to a National Institutes of Health Sometimes many man years of labor were lost and careers

were changed in failed attempts to duplicate a result. Forsponsored symposium at the Heart House with the remarkthat ‘‘Anyone who has read the literature in CF and says example, several years and literally thousands of rats (D.

Dearborn, personal communication) were invested in try-he isn’t confused, is confused.’’The first reported putative cellular defect was an off- ing to replicate the transport inhibitory factor effect re-

ported on rat parotid glands (171, 269). None of these re-shoot of observations in glycogen storage disease inwhich Danes and Bearn (63, 64) reported that fibroblasts ports has been substantiated rigorously. Some have been

refuted or seriously challenged (22, 38, 209, 258, 268). Acultured from CF patients developed significantly moremetachromatically stained granules than those derived very few have been courageously retracted (36).

Many of these reports most likely represent errone-from normal subjects. Later in the course of examiningthe effect of tobacco smoke on ciliary beating, Spock ous starts and premature conclusions that were supported

by the ease of publishing in a field without a reliable,(242) reported that serum from patients with CF causedincreased mucus release and induced a marked dyskinesia reproducible scientific point of reference. Often studies

of this period rested on an uncritical faith in publishedin the ciliary action of rabbit trachea when applied tothe luminal surface in vitro. The excitement gained much results. The renowned CF physician Harry Shwachman

once remarked ‘‘There have been more Chairmen of pedi-momentum when Bowman et al. (34) reported that theeffect could be duplicated using oyster cilia and Dogett atrics made from CF than from any other disease because

anything can be published’’ (personal communication). Asand Harrison (83) reported that it could be reversed byheparin and that a cationically charged protein was the a part of the CF story, these works may be most valuable

in emphasizing the importance of setting critical experi-‘‘factor’’ responsible for the effect.The idea that a circulating humoral factor was at the mental standards and in posting a warning of the pitfalls

that must accompany attempts to tie empirical pathologi-heart of the pathology gained still greater attention whenMangos and co-workers reported that sweat or saliva from cal observations to underlying physiological and biochem-

ical phenomena.CF patients applied to the luminal surface of rat salivarygland ducts (172) or human sweat gland ducts (173) inhib-ited active Na/ absorption in these epithelia. Rao and IV. THE MUCUS STORYNadler (202) suggested that a defect in arginine esteraseaccounted for these effects by elevating the concentra- It is now clear that abnormalities extend well beyond

fibrotic cysts in the pancreas and that several target or-tions of cationic amino acids. A large, heat stable macro-molecule in CF tears and stool fluids was reported to gans such as the sweat, lacrimal, and parotid salivary

glands do not produce ‘‘viscid mucus’’ (194). Nonetheless,stimulate excessive mucin release from the urn cell of theexotic marine invertebrate Sipunculus (10). no doubt because of the impressive histological findings

of accumulated mucus in the airways and to apparentThen, Wilson and co-workers (270, 271) reported thata unique protein band appeared in CF serum electropho- ‘‘mucous plugs’’ in other organs (31, 96, 277), the idea that

CF was a disease primarily caused by thick sticky mucusresed through a pH gradient to its point of isoelectricfocus. Lieberman and co-workers thought that an elevated to this day commonly pervades the thinking of physicians,

scientists, and patients alike. This perspective of the dis-protein with lectinlike binding properties that could bedetected via agglutination of red blood cells (163), but ease was entrained by observations in several exocrine

organs that appeared to be based on some form of abnor-which disappeared during menses (164), reflected the ba-sic defect. Katz and co-workers (223) reported that intra- mal mucus secretion or metabolism.


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A. Pancreas cells, but probably not more so than other chronicallyinflamed airways (207).

Cystic fibrosis patients with infected airways gener-Abnormalities in the pancreas made it easy for earlyally produced sputum that was thick and viscid, but itsinvestigators to conclude that the fundamental abnormal-physical properties were largely determined by the pres-ity was altered and/or increased in mucus production.ence of cellular and bacterial debris, especially DNA,Overt symptoms of pancreatic insufficiency did not ap-which made it impossible to define the nature of (or possi-pear untilú85–90% of the pancreas was lost (69, 99, 174).ble differences between) pure mucus secretions from na-Pancreatic insufficiency developed in utero in some pa-tive uninfected CF and control airways. Nonetheless, thetients and over a period of many years in others (259).gross appearance of the expectorant as well as analysesThe progression has been qualitatively estimated as in-reporting abnormally low water content (96% for CF vs.volving four (subjective) degrees of involvement (65): 1)98% for bronchiectasis) supported the notion that airwayeosiniphilic concretions in the lumen, 2) widening of themucus secretions were abnormally thick and ‘‘dehy-ducts with intra- and interlobular fibrosis and microcystsdrated’’ (137a, 185–187). Even now, the question remainsappearing to be due to ductal obstruction, 3) acinar andas to which occurs first, increased (abnormal) mucus se-ductal atrophy and disintegration accompanied by fattycretion or infection and inflammation (17, 137, 139). Un-infiltration, and 4) scar tissue surrounding isolated isletsdoubtedly, it is noteworthy that infections begin in theof Langerhans with total loss of acinar and ductal struc-small bronchioles before detectable loss of lung functiontures with increased incidence of insulin and glucagontakes place (66, 184, 251). Pertinent to the question, thereinsufficiency due to ‘‘strangulation’’ of the islets. The in-is very recent evidence from studies of a specially bredspissated plugs or concretions in the ducts were reportedCF-gene knockout mouse that changes in mucus in theto contain high concentrations of Ca2/ (151). In general,airway may precede disease (133). It remains significantthe caudal pancreas was found to deteriorate more rapidlythat despite prodigious efforts to define a chemical aberra-than the head, and although there is no bacterial infection,tion in mucins produced in CF, to date no unique composi-mild leukocytic infiltration was seen even in early stagestional differences have been defined (114, 159). However,of the destruction (71). The progressive loss of functionsome progress may be being made (56, 220) in redefiningwas highly variable, not an ‘‘all or none’’ phenomenon,early reports of alterations in the sulfation (29, 53) and inand in some cases seemed to occur almost ‘‘unit by unit’’sialylation and fucosylation of CF mucus (79). The earlyover extended periods as ducts were obstructed and acinirecognition that DNase had marked thinning propertieswere lost to ‘‘mucus blockage’’.on crude CF sputum (52) and the recent application of thisknowledge through the therapeutic use of cloned humanDNase to reduce sputum viscosity in vivo (189, 201, 232,

B. Airways 233) underscored the fact that viscosity was not solelydue to abnormal mucus. Thus the disease in the lung wasfrequently perceived as being caused by thick mucus, thatImpressions from the pancreas that CF was a disease

of abnormal mucus were strongly reinforced by lung pa- was clearly ‘‘abnormal’’ as compared with normal lungsecretions, but the more appropriate comparison is proba-thology. In young infants, even before significant anatomic

changes occurred, the appearance of bronchial mucus bly to mucus produced in other diseased lungs. Introduc-tory sentences such as ‘‘Abnormally viscoelastic secre-hypersecretion was the first notable evidence of a pulmo-

nary lesion (277). It remains an enigma that in contrast tions in CF cause impaired mucus clearance and persis-tence of bacteria within the lung’’ were and are commonto most other lung diseases, the upper lobes fell prey to

infection before the lower lobes, and like the pancreas, in the literature of CF. That is not to say that there is nochemical abnormality in CF mucus, but it seems that itthe lungs deteriorated over months or years seemingly

almost unit by unit. Frequent involvement of the airways was not widely recognized that such an abnormality wasstill far from being established and that other diseases,was consistently noted at autopsy as dilated bronchi with

tubular bronchiectasis. Bronchial lumens were filled with asthma and especially status asthmaticus (250), bronchi-ectasis without CF (176), pseudohypoaldosteronismpurulent exudates (4, 31, 93, 94). The tracheal bronchial

submucosal glands appeared normal in development but (117), Kartagener’s (immotile cilia) syndrome (221), andsome cases of Sjogren’s (autoimmune destruction of exo-frequently hypertrophied with dilated lumens that under

the microscope seemed to be filled with dense mucus crine tissue) syndrome (61), all produced sputum that is‘‘thick and viscid’’ or ‘‘dry,’’ but did not result in CF lung(207). Histochemical studies suggested that glands in

chronically infected patients produced more highly sul- pathology (112). Cystic fibrosis sputum was found to bealmost uniquely characterized by bacterial flora con-fated mucins, but probably not more so than non-CF, dis-

ease-related controls (154). Similarly, the airway epithe- taining a mucoid form of Pseudomonas aeruginosa thatproduced alginate. This protein, commonly used as a foodlium appeared to be excessively populated with globlet


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thickener, was once thought to be largely responsible for cosal fluid-filled cysts, but these changes apparentlyoccurred without inflammation (92). As many as 20%the viscosity of CF sputum (84, 85). Why the CF lung is

predisposed to this form of a ubiquitous bacteria remains of CF patients were observed to suffer cholelithiasis(gallstones), but it is most likely that this complicationas another mystery. Unfortunately, the complexity of spu-

tum, the nature of the infection, and the individual vari- was secondary to depletion of the bile acid pool be-cause of decreased digestive enzymes and increasedability of the inflammatory responses (137a, 138, 196) have

prevented unassailable comparisons of the composition stool (259).of mucus in different diseases.

D. Reproductive Tract

C. Gastrointestinal TractObservations in the female genital tract gave fur-

ther evidence of a disease of viscid mucus. InspissatedThe biliary tree, the gallbladder, and the intestine

mucus plugs were sometimes found obstructing thehave all been reported to show similar signs of increased

cervical canal (183). At times, the endocervix and cer-mucus or ‘‘thickened’’ mucus production. Clearly, one of

vix were found to exhibit polypoid masses presumedthe most dramatic and compelling demonstrations that

to contain inspissated mucus (86, 183). The gross qual-CF was a disease of ‘‘mucoviscidosis’’ derived from the

ity of the mucus was found to be thick, tenacious, andpathognomonic association between CF and meconium

of relatively low volume compared with controls (150).ileus. During the 1960s, sufficient records were accumu-

Cystic fibrosis women showed a significantly lower fer-lated to demonstrate that Ç10% of CF patients were born

tility rate thought to be due to the impediment thewith this condition (75, 216). In these infants, the small

thick mucus imposed upon sperm entering the cervicalbowel was blocked with a thick, dehydrated, rubbery,

canal (59).tarry, tenacious mucoid plug. The loss of pancreatic en-

The tissue most sensitive to CF in terms of embryo-zymes in utero undoubtedly gave rise to accumulations

logical development was the epididymal duct and vas def-of undigested proteins (e.g., albumin) which when mixed

erens. In most cases of CF, these structures were foundwith intestinal mucus produced the impervious, hypervis-

to be incomplete (129, 181). It was speculated, but notcid meconium substance. Microscopic examination of the

proven, that the atresia was due to blockage of the duct-surrounding intestinal epithelium showed dilated mucus

ules in utero (123, 155, 160). Most males with CF wereglands with prominent mucus cells along lumens that con-

found to be aspermic or hypospermic (123).tained a ‘‘stringy’’ secretion (31, 126, 255). It was takenalmost as a given that the mucus glands of the colonsimilarly demonstrated an increased proportion of mucus E. Salivary Glands

goblet cells (31), but a carefully executed morphometricstudy of colonic biopsies showed no significant differ- A few other morphological aberrations mainly in the

submaxillary and minor labial salivary glands also seemedences between goblet cell distribution in CF and normalsubjects. Differences in individual cell morphology be- consistent with abnormal mucus. These glands were re-

ported to contain mucous plugs and eosinophilic stainingtween CF and normal cells were attributed to the highlipid load associated with pancreatic insufficiency (179). mucus in their lumen (231), but it was not possible to

conclude that these observations were strictly unique toA more recent ultrastructure study of the small gut sug-gested that cytological abnormalities were associated the disease (161). Figures 1 and 2 offer an example of the

kinds of microscopic changes that have been observed inwith crypt cells where the highest concentration of cysticfibrosis transmembrane conductance regulator (CFTR) mucus-producing exocrine glands in the form of minor

salivary glands from CF and control subjects. Even thoughwas expressed (219).Liver cirrhosis was not usually encountered until these glands were not known to become infected or in-

flamed or to autodestruct by autolysis, dilated ducts withmuch later in life and then in only a few percent of pa-tients, but a large fraction of patients at autopsy exhibited thinner walls were outstanding features of the CF speci-

mens, but what appeared to be more darkly stained mate-‘‘focal biliary cirrhosis’’ in which focal zones of numerousbile ducts and tubules contain distinctly eosiniphilic, gran- rials, thickened, were present in the lumens of the ducts

of both glands.ular secretions that appeared to form plugs that causedductal dilation (72). Barbero and Sibinga (13) found that salivary glands

were significantly enlarged in CF, but this result was thenIn about one-third of autopsied cases, the gallblad-der was found to be hypoplastic and to contain thick interpreted to be due to the chronic administration of b-

adrenergic agonists (222, 230). Saliva from CF patientsor gelatinous mucus, viscid green bile, clear jelly, solidgreen bile mucus sometimes along with obliteration of demonstrated an abnormal color and turbidity, presum-

ably due to higher concentrations of Ca2/ and phosphatethe cystic duct. These bladders often exhibited submu-


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FIG. 1. Hematoxylin- and eosin-stained tis-sues from cystic fibrosis (CF) minor labial sali-vary glands. Note ducts are distended with rela-tively thinner epithelial walls (right arrow).Acini appear dilated also (middle arrow). Ironi-cally, substance in lumen (left arrow) for mostpart appears less ‘‘dense’’ and ‘‘thick’’ than innormal tissue in Fig. 2. Magnification,1200. (Mi-crograph courtesy of Dr. Walter Finkbeiner, Uni-versity of California, San Diego.)

(30, 169) that appeared to reverse upon administration of impact on understanding mucus abnormalities in CF.However, once it became appreciated that CFTR was aguanethidine (55). Pancreatic enzyme supplements were

thought to influence salivary composition as well (247). Cl0 channel, Al-Awqati and co-workers (1, 11) reportedthat normal acidification of intracellular vesicles in CFcould not be achieved and that posttranscription sialyla-

F. The Cell tion and fucosylation of mucous molecules would be de-creased and increased, respectively, due to the effect ofthe altered pH on the optima of the glycosylating enzymes.Even though CFTR, the gene affected in CF, was

cloned 10 years ago (134, 210, 215), the finding has little However, Verkman and co-workers (25, 229) found no

FIG. 2. Hematoxylin- and eosin-stained tis-sues from normal minor labial salivary glands.Walls of duct are clearly thicker (top rightarrow), and lumen diameters are significantlysmaller relative to comparable structures in CFgland. Acini are not dilated, and their lumensare hardly discernible at this level (left arrow);however, substance in lumens (bottom rightarrow) could be interpreted as being ‘‘thick-ened’’ compared with CF tissue submandibularglands (Fig. 1). These examples are providedto not only show tissue differences but also toillustrate ambiguity in interpreting ‘‘appearance’’of mucus in histological specimens as was com-mon in early pathology studies. Magnification,1200. (Micrograph courtesy of Dr. Walter Fink-beiner, University of California, San Diego.)


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TABLE 1. Electrolyte concentrations in control and CFdifferences in intraorganelle pH of CF cells and vigorouslysweatrefuted this observation and speculation.

Electrolyte Concentration, mM

V. THE ELECTROLYTE STORY Cl0 Na/ K/ Ca2/

Control 16 23 8 0.65Except for the sweat gland, most clinical distur-CF patients 99 101 15 0.83bances have been related to abnormalities that appeared

to be directly related to abnormally viscid mucus secre- Measurements are averages from more than 500 cystic fibrosis(CF) and 1,000 control subjects and should not be taken as fixed ortion. The three to five times normal elevation of NaCl inabsolute (see text). [Data from di Sant’Agnese and Powell (76).]the sweat of CF patients was long viewed as something

of an outlier in the etiology of the disease because thesweat gland was without significant mucus. Even Ander-

tolerance among troops sent to North Africa was attrib-sen was reluctant at first to believe that it could be im-uted to adaptations in sweating, he pursued excessive lossportant to the basic pathology of the disease (diof salt in the sweat as the most likely origin of volumeSant’Agnese, personal communication). A few investiga-depletion during the high heat stress (personal communi-tors tried to reveal similar electrolyte abnormalities in thecation). He began collaborating with Darling, Perera, andsalivary glands (27, 74, 170, 263). In general, the Na/, Cl0,Shea to prove it. Renal output and conservation of Na/and K/ concentrations in CF salivary secretions, more sowas within normal range. Nothing in the stool could bewith submaxillary and labial than with parotid glands,implicated. However, when sweat was collected and ana-appeared at somewhat higher concentrations than thoselyzed, the concentrations of both Na/ and Cl0 were greatlyfrom normal glands, but although Ca2/ was consistentlyincreased in sweat from CF patients (73). It was not possi-found to be about twice normal, the results have not al-ble to give strict concentration values for these ions inways been statistically significant (27, 194). If there areeither CF or normal subjects simply because the fractionaldisturbances in tear glands, they are not large, probablyabsorption of salt from the secreted sweat varied enor-because these glands secrete a relatively unmodified iso-mously with secretory rate, age, circulating aldosteronetonic fluid (32, 74, 235). Throughout the history of CF, nolevels, acclimatization, drugs, and any number of less ob-exocrine secretion or physiological function has providedvious factors (167, 236). For example, in salt-depleted nor-as clean a discriminator between normal and CF subjectsmal individuals, fractional absorption ranged from almostas has sweat.90% for sweating at low sweat rates to only a few percentUndoubtedly, the overt lack of any clinically apparentin some subjects surfeiting on sodium and sweating atrenal disturbance undermined early thinking of CF as avery high rates (personal observations; Refs. 81, 90, 91,disease of a generalized disturbance in electrolyte trans-168). After analyzing samples from hundreds of patientsport (76). The CFTR was found recently to be expressed(76, 236), however, it became evident that two relativelyin the human kidney (62, 178), but there was little evi-distinct zones separated normal from CF pediatric sub-dence of renal compromise. No morphological abnormali-jects (Table 1). In normal subjects, sweat concentrationsties have been observed (177). There is an increased unex-of salt fell below Ç60 mM, and in CF, sweat concentra-plained clearance of several drugs, especially aminoglyco-tions rose above that value with almost no overlap (76,sides (9, 97, 128, 157, 274). A few reports of other236).3 In an extensive study, the average sweat electrolytefunctions such as reduced concentrating and diluting ca-values for siblings and parents of CF patients were foundpacities (210) or altered glomerular filtration rate (121,to be slightly, but significantly, increased over the normal243) were either not followed up or remained controver-population (76). A few years later, Shwachman and Mah-sial (241).moodian (236) refuted these results in putative heterozy-gotes in another extensive report.

A. Sweat Gland Sweat had much value as a parameter in differentialdiagnosis, but it was cumbersome to collect and difficultto obtain without serious evaporative losses. Some earlyAs noted in section II, the first indication that CF

extended beyond an apparently abnormal mucus ap- attempts to place the subject in a body bag to obtainenough sweat for analysis proved that this approach pre-peared when di Sant’Agnese set out to determine the etiol-

ogy of volume (salt) loss in Andersen’s patients afflicted sented a high risk for fatal hyperthermia. In the late 1950s,Gibson and Cooke (104) seized the idea of stimulatingby the heat wave that hit the northeast United States in

the summer of 1948.2 Noting that the development of heat localized sweating by electrophoresing (iontophoresing)

3 The overlap is much greater with adults, presumably because of2 One cannot help but wonder about the course of events had airconditioning been common then. the increased secretory capacity relative to its absorptive capacity.


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the positively charged secretagogue pilocarpine into an In 1981, Knowles et al. (140) reported that the electro-negative potential across the nasal airway in CF was sig-area of skin of Ç20 cm2. The area was then hermetically

sealed under taped plastic, and after Ç30 min, the sweat nificantly larger than normal. This report along with thefacts that NaCl absorption was inhibited in CF sweat ductsabsorbed into the gauze was weighed, eluted, and ana-

lyzed for concentrations of Cl0.4 The protocol has become and that Na/ was relatively more absorbed than Cl0 gaveme the idea that the basic defect in the CF duct had tointernationally used and accepted as almost mandatory

for the diagnosis of CF (158). Only a few other very rare be due to anion impermeability (192) and not to defectiveanion exchange (191) (as I had been thinking earlier basedconditions created false-positive results. Among them

were Addison’s disease (82), pseudohypoaldosteronism on chemical measurements) or to increased Na/ absorp-tion as was postulated for the airway (140).6 However,(117), hypothyroidism (60), and malnutrition (48, 57, 175).

A few isolated cases diagnosed with CF have been re- the fluid volumes involved in microperfusion of singleducts were far too small and the specific activity of Cl0ported with sweat Cl0 concentrations in the normal range

(67, 244), but it is not clear whether these observations too low to accurately measure isotope fluxes to prove Cl0

impermeability. In microperfused isolated segments of ab-are true exceptions with an unknown physiological com-pensation or whether normal physiological parameters sorptive ducts from CF and normal subjects, I found that

the average spontaneous transepithelial potential of nor-that have not been accounted for are at play.Numerous studies were conducted over the next de- mal sweat ducts was Ç7 mV, whereas in CF ducts it was

Ç75 mV (193). In the meantime, on the suggestion ofcade that firmly established the validity of the sweat test(70, 76, 105, 158, 236), but in the course of these studies, Maurice Burg, I found that we could demonstrate the

anion impermeability electrophysiologically by ion substi-several observations were also noted that would formthe basis of, or provide helpful insights into, the future tution and showed that replacing Cl0 with the relatively

impermeable SO204 anion in normal, microperfused ductsunderstanding of the physiology of CF. First, almost con-

temporary with di Sant’Agnese’s observations, the ‘‘two- raised the value of lumen negative potential to about equalthat of the CF duct, whereas the maneuver had muchstep’’ theory of exocrine fluid formation was published

(227). The theory predicted that exocrine fluids were first less effect in CF ducts (193). Corroborating results wereobtained a little later in single glands in vivo (23, 198).secreted as an iso-osmotic fluid in the secretory or acinar

structure of the gland and secondarily were modified in Because the defect was expressed in the morphologicallynormal sweat gland, it appeared to be independent of anycomposition as they passed through the ductal or tubular

network of the gland before being excreted.5 Direct ultra- secondary pathological or degenerative consequences dueto infection, inflammation, or mucus disturbances thatmicro sampling from the gland (224–226), histological

cryo-osmometry (237, 239), and linear extrapolation (48, often occurred in other target tissues that secreted macro-molecules. Thus this property not only provided a physio-80) were important approaches that verified the theory

and answered the initial question of whether CF sweat logical explanation for the high salt content in CF sweat,but it also provided the first description of a basic, cellularwas higher in concentration due to a primary secretory

defect that secreted fluid at hypertonic concentrations or defect that has since proven to be uniformly inherent inCF affected cells.due to an absorptive defect in which ductal electrolyte

absorption failed in CF. It was also noted that sweat Na/, Abnormalities in adrenergic control in CF had beensuspected for several years (12, 246). Schwarz and Sut-but not Cl0, in CF dropped as expected in response to

increased levels of circulating aldosterone (78). Slegers cliffe (228) suggested that the uptake of salt from the ductmight be influenced with adrenergic agonists. But not until(238, 239) also observed that the ratio of Na/ concentra-

tion to Cl0 concentration in normal sweat was almost 1984 did Sato and Sato (217) find a fundamental corollaryof the Cl0 impermeability defect in fluid secretion, alsoinvariably ú1.0, whereas in CF sweat, it was almost al-

ways õ1.0. Both of these observations hinted that Cl0 in sweat glands. They found that the secretory activity ofCF sweat glands was almost completely insensitive to b-was less effectively absorbed than Na/. Schulz and

Fromter (225) noted in three patients with CF that the adrenergic stimulation.7 The insensitivity was later shownto be due to the fact that in the sweat gland b-adrener-electrical potential associated with sweat production on

the skin was more electronegative than normal and even gically stimulated fluid secretion was coupled to the sameCl0 channel that was altered in salt absorption (195). Thesuggested that this could only occur if Cl0 were less per-

meable than Na/, but these results were neglected and Cl0 conductance in absorption was also under b-adrener-apparently were not mentioned again until pointed out by 6 I felt some empathy with di Sant’Agnese because I presented thisa reviewer of my manuscript some 14 years later (193). idea in 1982 at a Cystic Fibrosis symposium where I too was the last

speaker of the evening, and although present, most of the audience wasasleep.4 The differences from normal in Cl0 concentration are larger than

differences in Na/ concentration. 7 b-Adrenergic innervation serves no known physiological func-tion; thermoregulatory sweating appears to be purely cholinergic and is5 Some lower vertebrates, but not mammals, can secrete hyper-

tonic fluids (190). not abnormal in CF.


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TABLE 2. Comparison of electrolyte composition and rate of pancreatic secretions collected from the duodenum of

control and pancreatic sufficient CF patients

Electrolyte Composition, mMVolume, Trypsin, Protein,

Cl0 HCO03 Na/ K/ mlrkg01

rh01 mg/ml mg/ml

Hadorn’s study

Control subjects (n Å 10–30) 60 (258) 75 (258) 107 (73) 11 (73) 4.7 (115) 220 (115)CF patients (n Å 10) 86 (115) 19 (115) 119 (73) 9 (73) 0.8 (115) 515 (115)

Kopelman’s study

Control subjects (n Å 9) 81 45 142 8 9.7 2.75CF patients (n Å 9) 77 39 151 8 4.9 5.6

Reference numbers are given in parentheses. Data for Kopelman’s study are from Kopelman et al. (149).

gic, cAMP-dependent control (199, 200, 206, 267). Chloride of Vater and collect relatively pure pancreatic secretionsimpermeability and insensitivity to activation by protein also during in vivo pharmacological stimulation with intra-kinase A has now become the signature of expression venous pancreozymin/secretin. Although Kopelman et al.of CF in the single cell, whether native, transfected, or (148) did not find significant differences in electrolyte con-cultured.8 Although overlap was too great to permit detec- centrations (Table 2), both studies concluded that macro-tion of heterozygotes on an individual basis, the average molecules were abnormally concentrated and thatsweat rate of the heterozygote population in response HCO0

3 and volume outputs were abnormally low in CFto a b-adrenergic challenge still remains one of the few, patients. The fact that secretin-stimulated HCO0

3 secretionperhaps the only, physiological parameter that showed was mediated by cAMP-dependent protein kinase and thatintermediate expression of the gene in vivo (18, 218). the pancreas was so adversely affected in CF revealed

that Cl0 impermeability has a pronounced negative effecton exocrine HCO0

3 transport. This effect still remains asB. Pancreas

one of the most important but neglected and poorly under-stood aspects of pathophysiology in CF. With the recogni-Untreated, the large majority of CF patients sufferedtion that fluid secretion was inhibited, the explanation forfrom severe malnutrition that was attributed to the losspancreatic deterioration no longer required ductalof pancreatic parenchyma and the concomitant loss ofblockage by abnormally viscid mucus. Without sufficientdigestive enzymes. Some questioned whether the defectfluid and HCO0

3 , digestive proenzymes stagnated and acti-in sweat NaCl might be due to poor nutrition. About 10% ofvated prematurely in the pancreatic ducts. The results,diagnosed patients escaped pancreatic insufficiency (71),nonetheless, were the same: microautolysis, focal injury,which we now know is due to different genotypes (135,inflammation, infiltration, calcification, plugged ducts, fi-152). Although the gross and microanatomy of pathologi-brosis, and scarring until the entire parenchyma of thecal changes in the pancreas were obvious from the outset,pancreas, like the lung, but from a seemingly differentit was not until the late 1960s that Beat Hadorn recognizedcause, was destroyed.that fluid and electrolyte secretion, like electrolyte ab-

The first fundamental work on the molecular proper-sorption in sweat, was also abnormal in CF. In 10 patientsties of the Cl0 conductance affected in the disease alsowho did not exhibit overt pancreatic insufficiency, se-arose from the pancreas. The earliest attempts (101, 264)creted volumes and HCO0

3 content were markedly de-to use patch-clamp techniques to show the molecular de-creased in CF while Cl0 and digestive enzyme concentra-fect through Cl0 single-channel activity encountered se-tions were increased (115, 116, 125) (Table 2). Most CFvere controversy. Several investigators, most of whom didpatients were relatively refractory to secretin stimulationnot publish (J. Hanrahan, J. Wine, J. Bijman, and R.but responded to pancreozymin. These early observationsGreger), were unable to repeat the published results thatwere the first evidence that like the sweat gland, only onea nonlinear rectifying Cl0 channel could be activated byof at least two mechanisms of fluid secretion involved inb-adrenergic agonists, as required for the affected channelpancreas was seriously impaired.in CF (153). After 2 years of controversy, using an organAbout 15 years later, Kopelman et al. (149) used anculture system of human pancreatic ducts developed byenteric double-ballooned catheter to isolate the ampulaAnn Harris, Argent et al. (109) found two types of Cl0

channel activity: one with outwardly rectifying chord con-8 Protein kinase A is the most established intracellular mediatorof CFTR Cl0 conductance, although other pathways may be involved. ductances ranging fromÇ19 to 53 pS was seen rarely and


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TABLE 3. Concentrations of electrolytes in airwaywas activated by strong depolarization and another withsurface fluids from two studiesÇ4–7 pS linear conductance was abundant and activated

by cAMP/protein kinase A. These results were rapidly con-Electrolyte Concentration, mM

firmed (21, 58, 120, 273), and the rectifying channel wasCl0 Na/ K/shown not to be correlated with CF (262).

Joris’ study

CF patients 121 129 23C. AirwaysControl subjects 82 84 29

While searching for evidence that electrolyte trans- Knowles’ study

port in the airways was important for host defense,CF patients 76 86 24

Knowles et al. (140) included a few CF patients in his Control subjects 80 88 22cohort of asthmatic and heart disease subjects. He discov-ered that the electrical potential associated with the nasalmucosa of CF patients was significantly more electronega-

spontaneous potential was read, Na/ conductance wastive with respect to blood than with all other subjects.blocked with amiloride, the mucosa was challenged withThis finding in 1981 provided the first physiological linkisoproterenol, a b-sympathomimetic agonist, and Cl0 wasbetween the lung, the pancreas, and the sweat gland (140).removed from the perfusate. The response of the electri-The common link was not mucus, but electrolytes. Al-cal potential to amiloride was two- to threefold larger, butthough it was not rapidly appreciated, the finding allowedthe response to b-adrenergic challenge and Cl0 substitu-the possibility that problems with mucus were due not totion was significantly smaller or nonexistent in CF sub-abnormalities with its synthesis or composition, but tojects; all effects were consistent with inherent Cl0 imper-the fluid environment into which mucus was secretedmeability in CF.(195, 196). In CF subjects, the spontaneous electrical po-

With the recognition that CF was a disease of electro-tentials measured across respiratory mucosa were aboutlyte transport came a renewed concern regarding abnor-twice normal (53 mV in CF vs. 25 mV in healthy controlmalities in the composition of the airway surface fluidssubjects) with no overlap in values. The difference in elec-(ASF). Earlier attempts to gain insights from sputum andtrical potential between CF patients and normal subjectslaryngeal secretions collected from CF patients and con-persisted down the airways as far as the airway lumentrol bronchiectatic subjects indicated that the solid con-could be accessed, although the absolute values becametent of these fluids were increased (Ç10 vs. Ç5%) andsmaller so that in CF segmental bronchi the average po-that Na/ and Cl0 concentrations were lower, respectivelytential difference was 32 mV (CF) vs. 15 mV (normal) in(176, 186, 187). However, these results probably did notbronchi (145), lumen negative relative to blood. In thereflect the actual physiological values of the ASF in vivopresence of amiloride, a diuretic known to block epithelialbecause these secretions were badly contaminated withNa/ conductance, the transepithelial electrical potentialdebris from infection and inflammation. Unfortunately, itfell in both groups to about the same level (Ç5 mV).has proven to be extremely difficult to collect and analyzeKnowles et al. (140) postulated that in the airways, the‘‘pure’’ ASF from the airways because it normally existsincreased potential found in CF was due to significantlyin an extremely thin layer estimated to be from 10 to 50accelerated active Na/ absorption from the airway surfacemm in depth. More recent studies reporting the composi-fluids in the lumen. This hypothesis was appealing be-tion of salt in the fluid in the large airways are inconsistentcause it presented a mechanism (dehydration by exces-for CF. During bronchoscopy, samples were collected onsive fluid absorption) by which abnormally thick mucusfilter paper placed in the ASF on the airway wall. In addi-could be formed in the airways (33). The same Cl0 imper-tion to the difficulty of analyzing such small samples freemeability defect previously described in the sweat ductof evaporative and gravimetric errors, even these direct(192, 193) was then found in the airways (142, 144). So-sampling techniques suffer serious potential artifacts aris-dium absorption was not enhanced but clearly decreaseding from the invasiveness of the method and the respon-in the sweat duct, and it is not clear that Na/ transportsiveness of the epithelium to disturbances (Table 3).is affected in the pancreas or intestine. Neither the mecha-

As of this writing, the airway continues to build CFnism of enhanced Na/ absorption in CF airways nor itshistory. Recently, Smith et al. (240) reported that primarylink to the genetic defect has yet been explained.cultures of CF airway epithelial cells grown at an air inter-From these initial observations, a new clinical proce-face were compromised in their ability to resist bacterialdure using the potential differences measured across theinfections with respect to their normal counterparts. Theynasal mucosa was developed to assist in the diagnosis offurther showed that reducing the salt concentration ofCF (141). The potential was measured while the nasal

mucosa was perfused with different solutions. After the fluid collected from the CF epithelium restored bacteri-


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cidal activity and raising the salt concentration of the fluid enterocyte cells. Several other alterations in the gastroin-testinal tract have been reported, but clear abnormalitiescollected from control epithelia removed its bactericidal

activity. Similar results were reported for CF and control have been difficult to establish (110).Ironically, it now seems that these abnormalities incells grown on tracheal xenografts in immunodeficient

mice (107). Analysis of the salt concentrations in the ASF the gut may be the most likely cause for the high incidenceof some CF mutations, notably DF508. Earlier, the selec-over primary cultures supported the conclusion that, like

the sweat gland, the NaCl concentration in CF ASF was tive pressures thought to be responsible for the high genefrequency of CF were numerous and almost completelysignificantly elevated (276). These results may strengthen

the possibility that abnormalities in the composition of speculative (147, 188, 245). When it became clear that allthree target organs, the sweat gland, the pancreas, andthe airway fluid as opposed to inherent abnormalities in

the composition of airway mucus was the component that the lung, were products of electrolyte transport distur-bances, I suggested altogether too narrowly that the selec-predisposes the CF lung to airway disease.tive advantage of the CF heterozygote might be in theintestine and related it to surviving cholera infections

D. Intestine (192). I did not realize that cholera did not strike Europeuntil the 19th century, obviating this plague as a selectivepressure. However, I later learned that that diarrheal dis-Work on the electrolyte abnormality in the gut was

slow to evolve probably because of an early prevalence eases caused by ubiquitous organisms such as enterotoxicEscherichia coli (197) have affected humans for longof the idea that malabsorption in CF was essentially due

to pancreatic dysfunction. In 1985, the first transport ab- enough period to conceivably constitute the selectivepressure. Proof of the notion has not been forthcoming,normality in the CF intestine was reported as an enhanced

rate of glucose uptake in vivo (100), which was proposed but CF patients have been found to be refractory to acuteexposure to Sta (heat-stable E. coli enterotoxin) in vivoto be due to smaller diffusional barriers. The finding was

later confirmed in vitro and shown to be associated with (108). The survival of transgenic CF mice exposed to chol-era toxin perhaps provided the strongest support of thesignificantly higher nutrient-stimulated short-circuit cur-

rents in CF (15, 118, 254). Additional electrolyte transport hypothesis (102).disturbances in the CF intestine arose from observationsthat jejunal biopsies from CF patients in contrast to nor-

E. The Gene and Cellmal subjects failed to respond with a significant increasein lumen to serosal positive current when stimulated withPGE2, dibutryl cAMP, acetylcholine (252, 253), or the Ca2/ In addition to being a dramatic achievement in sci-

ence, the search for the CF gene was one of the greatestionophore A-23187 (180). Consistent results were ob-tained in the ileum, colon, and rectum (20, 24). The in successes in CF research. Because there was no known

gene product and only Cl0 impermeability, a membranevivo intraluminal potential in the jejunum when perfusedwith Cl0-free solution was significantly more positive in function property, was known to be characteristically al-

tered, the gene had to be found without a chemicalCF than in control subjects, whereas theophylline stimu-lated net fluid secretion in control, but not CF, subjects marker. The alternative approach was positional cloning,

but only the assumption that the mutated gene had to be(254). Both the increased Na/-dependent nutrient uptakeand the depressed secretory responses of the CF gut were expressed in the sweat gland, where Cl0 impermeability

had been demonstrated, linked the patient phenotype tocompatible with an inherent loss of Cl0 conductance. Theabsence of a normal anion shunt in the apical membrane the positional cloning effort. Before 1985, the location

of the CF gene in the human genome was completelyshould increase the apical membrane Na/ gradient of theenterocyte in CF, thereby producing larger currents when unknown. Eiberg et al. (89) observed that a polymorphic

serum paroxonase was inherited with high frequencynutrients were present. As for diminished secretion, anyagonist that required the CFTR Cl0 channel in the apical (linked) with the CF gene. In the same year, Tsui and

co-workers (146, 257) used another restriction fragmentmembrane would necessarily fail to elicit a response ifthe channel were missing or nonfunctional. linked polymorphism (RFLP) marker to localize the gene

to the long arm of chromosome 7. Thereafter, the raceThere was accompanying evidence of pH aberrationsin the CF intestine. Postprandial contents in the early gut was on to find the gene. Its localization was narrowed to

within 1–2 1 106 base pairs using RFLP. The most vigor-remained at a lower pH (õ6.0) for significantly longerperiods of time than in that of control subjects (106, 211, ous competition was between Williamson’s group in Lon-

don and Tsui’s group in Toronto. It was rumored that Bob275). These effects may be simply the result of diminishedpancreatic HCO0

3 secretion but could also, like the pan- Williamson had the gene, but the lead did not hold. In1985, in a stunning trilogy of papers published simultane-creas, reflect a compromised ability of the intestine to

secrete HCO03 as a function of lost Cl0 permeability in the ously (134, 210, 215), the Toronto group including Jack


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Riordan’s laboratory supported by Francis Collins’ group would require enhancing expression and delivery of themutant protein to the membrane. Still, direct proof thatat Michigan reported the cloning of the gene for CF. The

gene was conserved throughout numerous species, indi- CFTR was a Cl0 channel was lacking. It remained possiblethat it was a regulator, not a channel itself. The first evi-cating its evolutionary importance; it was predominantly

expressed in the target organs affected by the disease dence that CFTR was a channel was presented by mutat-ing lysine at position 95 and 335 to glutamate and showingincluding cells from the sweat gland. The cDNA hybridiz-

ing to the candidate gene sequences from the CF locus that this change reversed the anion selectivity sequenceof the channel (7). Although Greger and co-workers (122)were isolated from libraries constructed from sweat gland

mRNA from CF patients and unaffected subjects. Se- refuted these results, it is difficult to imagine how suchalterations would cause changes in permselectivity if theyquence comparisons between initial partial cDNA from

the two sources revealed the absence of three nucleotides were not directly involved with conducting the anion.Bear et al. (16) provided the first direct proof that CFTRin the patients even before the full-length sequence was

obtained, which was ultimately necessary to predict the was a Cl0 channel by reconstituting CFTR protein intoartificial membranes and demonstrating cAMP/ATP-de-protein product structure. The subsequent finding that

this DF508 deletion was present in almost 70% of the pendent channels with the same properties as those pre-viously described for CFTR wild type expressed ex vivochromosomes from patients diagnosed with the disease

provided assurance that the correct gene had been found. in patch clamping.We pass over an avalanche of data provided subse-They named its protein product CFTR, suggesting that it

might not be a Cl0 channel itself, but that it might regulate quently by patch-clamp and whole cell electrical re-cording techniques. The principal results of which de-Cl0 channel activity (210).

The next important step was to express the gene in scribed an ion channel with an anion selectivity of NO3

ú Br ¢ Cl ú I ú gluconate and a nonrectifying conduc-living cells. At first, great difficulty was encountered increating a full-length cDNA that could be propagated in tance of Ç6–8 pS (58, 109, 131, 273). The channel was

primarily activated by cAMP-dependent protein kinasebacteria and expressed in cells. Gregory et al. (111) clev-erly surmised and validated that a cryptic region in exon (58, 214, 248, 265). Activity was ATP dependent (6, 200),

and ATP hydrolysis was required for channel opening6 of the CFTR cDNA induced a transcript that was toxicto the host bacteria. When they inactivated the promoter, and for channel closing as well as a nonhydrolyzed li-

gand (14, 113, 205, 256).the bacteria were able to carry plasmids containing CFTRcDNA. This important conclusion was fundamental to de-veloping cell lines expressing the gene and its mutants.

VI. WHICH STORY TO BELIEVE?Using a low copy number plasmid, Welsh and co-workers(208) first corrected CF in cells ex vivo by transfecting aCF cell line with normal cDNA. The approach was quickly What does CF history show us in the past half cen-

tury? It is clear that CF involves electrolyte transport ab-improved upon by using mutated E. coli in which produc-tion of the toxic component was suppressed (54, 87). With normalities in all affected epithelial tissues and that mu-

cus secretion is or becomes enhanced in some tissues butthe advent of cell lines capable of expressing CFTR andits mutants came Seng Cheng’s startling discovery that not in others. It is clear that mutations in CFTR directly

affect Cl0 permeability and likely have other effects eithermutations in the putative nucleotide binding folds pro-duced proteins that were incompletely processed or de- directly or indirectly on Na/, HCO0

3 , and probably otherion distributions. However, the effect on mucus remainsgraded before reaching the membrane. The hallmark of

this event was failure to glycosylate asparagine residues unclear. Admittedly, I have been biased for many yearsthat the basic defect probably does not directly involveat positions 894 and 900 of the CFTR protein, which was

readily monitored from the electrophoretic mobility shift both electrolytes and mucus, largely because it seemedsuperficially, at least, incompatible with my obsolete edu-associated with the extension of the N-linked oligosaccha-

ride chains elaborated in the Golgi (54). Kartner et al. cation in the ‘‘one gene, one protein’’ concept. The sweatgland offers no support for a defect in mucus production(130) then showed that DF508 CFTR did not reach the

membrane in native CF epithelia. However, equally sur- or synthesis. The luminal blocks and concretions of thepancreas can probably be explained as the result of focalprisingly when mutant CFTR such as DF508 did reach the

membrane, it exhibited reasonably good function as a Cl0 stagnation of autolytic enzymes as discussed in sectionIVB. It has been tempting to say that the ‘‘abnormality’’ ofchannel (88, 162). Shortly later, Welsh and co-workers

(68) also discovered that these mutants were thermosensi- mucus in the airways is simply the result of infection andthat the apparently abnormal increase in viscosity is duetive, and growing cells at reduced temperature enhanced

processing and allowed expression of CFTR Cl0 channels. to the presence of DNA or to relative dehydration. Cer-tainly, a number of related anecdotal observations madeThese findings generated new thinking about therapeutic

approaches. They suggested that correcting the defect on very young infants as well as on adults dying of nonres-


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piratory causes report the presence of multiple casts and sporadic events seems to fit well with the disease process;that is, neither of the two organs that completely fail inplugs in very small airways that conceivably could pre-

cede infection. The lack of normal, cyclical changes in CF, the lungs and the pancreas, do so suddenly. Rather,there is progressive destruction (sometimes enduring de-the viscosity of vaginal fluids during the menstrual cycle

and the occasional mucus plugs encountered in the female cades) of the organ unit by unit as it were. In the airways,the sporadic insults that continually occur with debrisCF cannot be easily explained by infection either. In addi-

tion, the submucosal glands of the large airways and the that is deposited from inhaled air push isolated units be-yond the threshold of homeostasis to produce a localized,intestine have been described repetitively as containing

thick, stringy, or viscous mucus. It also seems unlikely destructive spiral of mucus release, accumulation of de-bris, and inspissation of ineffective mucus with subse-that these results are directly due to infection. Still, the

evidence that the mucus per se in these glands is abnormal quent loss of function. We know too little about variationsin acinar and lobular functions in the pancreas to be ablein CF is weak. We may surmise that the ‘‘apparently’’

abnormal mucus might well be a normal response to an to suggest what precipitating factors initiate focal stagna-tion in ductules, but it seems likely that such variationsunderlying challenge, but it is not possible to dismiss the

mucus condition in CF as simply due to chronic infection. exist and that beyond a point, localized flow and probablypH, becomes too low to provide proper clearance of theThere are fundamental tissue phenomena that may

help in integrating these events, at least from my armchair. unit before proteolytic enzyme activation and autolysisoccurs. Because a few percent of CF males are fertile, theFirst, one of the most primitive defenses that epithelia

offer to challenges from the outside world is mucus re- vas deferens may follow the same pattern, possibly againdue to problems in HCO0

3 and pH. Without further knowl-lease. Irritation of an epithelium results in hypersecretionof mucus. If mucus secretion is increased in CF and not edge of the processes incumbent with its development,

we cannot know which potentially destructive elementsinherently constituitively increased or a function of infec-tion, some form of ‘‘irritation’’ of the epithelium seems might be present, but stagnant lytic enzymes in the semi-

nal fluid seem likely to lead to the same course seen inlikely to be present. Changes in the composition (and/orvolume) of the epithelial surface or luminal milieu should the pancreas. The intestine and other organs largely es-

cape this tirade of deterioration because there seems toinherently accompany the physiological defect in electro-lyte transport in CF. Because epithelia are skilled in de- be few, if any, destructive elements such as highly concen-

trated digestive enzymes in the pancreas or intensely in-tecting surface or luminal changes, I ask if the defectivechanges in themselves could constitute a chronic irrita- flammatory agents in the airway to damage the epithelia.

The sweat gland escapes structural alterations for at leasttion to target epithelia. The defect would be even moreaggravating if CFTR has functions that extend beyond two reasons: 1) physiological sweating is driven by a se-

cretory mechanism that is not affected in CF so that therecreating a Cl0 conductance, especially if it either directlyor indirectly impedes the movement of other ions such is no reduction in flow, and 2) the composition of its

product, sweat, normally varies over a wide range thatas HCO03 , where the pancreas has tried so emphatically

to teach us for years that the transport of the two ions includes the composition of sweat produced in CF so thatthe composition in the CF gland lumen is still within amust be coupled. In addition to potentially constituting a

chronic irritation and provoking increased mucus secre- well-tolerated physiological range.Which story is correct? Neither a consideration oftion, alterations in composition also present an abnormal

environment into which mucus secretions are released, mucus nor of electrolytes alone is likely to present thewhole truth. Both must be interdependent and interre-leading to a second, related phenomenon: physical prop-

erties of mucus are intrinsically dependent on their envi- lated. History has not yet resolved where and why theyoverlap to produce the complex path that witches dubbedronment. The viscoelastic properties of macromolecular

polyelectrolytes such as mucus are critically sensitive to as a curse and that we in science still struggle to find.Until we chart that path, we may wander the Wild Westionic strength and pH (165, 166, 249). Alterations in the

ionic strength, divalent ion concentration, or pH are cer- with the stark possibility that even now we have not yetconsidered the true reason that CF is a disease.tain to affect the physical status of the mucus, which

again, in itself, may contribute to irritation to create avicious cycle. The facts that in nature mucus and HCO0

3REFERENCESsecretion are often coupled and that there is clearly a

defect in HCO03 movements in CF suggest that this aspect

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J. Exp. Biol. 172: 245–266, 1992.more important, to the clinical disease than Cl0 imperme-2. ALHADEFF, J. A., B. C. POLLACK, R. L. HOPFER, AND D. S. HOLS-ability, if indeed the two can be separated.

CLAW. Serum neutral alpha-D-glucosidase from patients with cysticThe possibility that this inherent weakness in the epi- fibrosis and chronic pulmonary disease. Pediatr. Res. 19: 171–174,

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4. ANDERSEN, D. H. Cystic fibrosis of the pancreas and its relation compartment of CFTR-expressing fibroblasts and T84 cells. Am. J.

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