Electrolyte
transport
in the epithelium #{149}
.
of normal CAROLE
and cystic
fibrosis
lung
of pulmonary
segments
1
M. LIEDTKE
Cystic Fibrosis Center, Department of Pediatrics at Rainbow Physiology and Biophysics, Case Western Reserve University,
The epithelium of pulmonary segments from trachea to aveoli actively transports electrolytes and allows osmotic movement of water to maintain the ionic environment in the airway lumen. Models of airway absorption and secretion depict the operation of transporters localized to apical or basolateral membrane. In many epithelia, a variety of electrolyte transporters operate in different combinations to produce absorption or secretion. This also applies to pulmonary epithelium of the large airways (trachea, main-stem bronchi), bronchioles, and alveoli. Na absorption occurs in all three pulmonary segments but by different transporters: apical Na channels in large airways and bronchioles; Na/W exchange and Na channels in adult alveoli. The Na channels in each pulmonary segment share a sensitivity to amiloride, a potent inhibitory of epithelial Na’ channels. Fetal alveoli display spontaneous C1 secretion, as do the large airways of some mammals, such as dog and bovine trachea. Cl- channels differ in conductance properties and in regulation by intracellular second messengers, osmolarity, and voltage mediate stimulated C1 secretion. Electroneutral carriers, such as NaCl(K) cotransport, C1/HCO3exchange, and Na/HCO exchange, operate in large airways and alveoli during absorption and secretion. Abnormal ion transport in airways of cystic fibrosis (CF) patients is manifest as a reduced Cl- conductance and increased Na conductance. Isolation of the CF gene and identification of its product CFTR now allow investigations into the basic defect. Intrinsic to these investigations is the development of systems to study the function of CFTR and its relation to electrolyte transporters and their regulation.-Liedtke, C. M. Electrolyte transport in the epithelium of pulmonary segments of normal and cystic fibrosis lung. FASEBJ. 6: 3076-3084; 1992. ABSTRACT
Key Words: epithelia ion transport phosphoinositides cystic fibrosis
channel
cotransport
poly-
PULMONARY EPITHELIUM, IN COMMON with other epitheha, absorbs and secrets ions to regulate the environment of the lumen of the organ. Inherent in this process is the vectorial movement of the most common electrolytes sodium and chloride with subsequent osmotic flow of water across the epithelium. Studies with epithelia from a variety of organs indicate the operation of diverse electrolyte transporters. In the intestine and kidney, different combinations of transporters operate in different segments to mediate overall absorption or secretion of fluid. This may also apply to pulmonary segments that range from nasal passages to alveoli. Evidence for this hypothesis comes from reports showing the many types of transporters that operate in one or more pulmonary segments (Table 1).
Babies and Children Hospital Cleveland, Ohio 44106, USA
and Department
of
This review focuses on major features of electrolyte transport required to achieve overall Na absorption or Cl secretion in pulmonary segments. A focal point for investigators is understanding how the airways from trachea to alveoli elaborate, secrete, and regulate a fluid layer that coats the apical surface of epithelial cells. The production of respiratory fluid involves the movement of ions and water across the airway epithelium from the submucosa to the lumen. This direction of flow defines the fluid movement as secretion. Driving the fluid movement is a Cl conductance in the apical membrane of airway epithelial cells. Figure 1 depicts a general model for C1-driven secretion in airway epithelium. Transepithelial C1 secretion requires the activity of four transporters, one localized in the apical membrane and three in the basolateral membrane. A basolateral NaCl(K) cotransporter mediates the uptake of Cl from the submucosa coupled to the entry of Na as it moves down its electrochemical gradient. Coupled uptake with Na provides the energy for Cl- entry. In some epithelia, cotransport with K doubles the number of Cl- entering the cell during expenditure of the same amount of energy from the Na electrochemical gradient. Na is pumped from the cell by the basolateral ouabain-sensitive Na,K-ATPase and then diffuses to the lumen across the tight junctions to maintain electroneutrality. K exits the cell at the basolateral membrane through a Ca2-sensitive channel. Rapid progress in identifying and characterizing electrolyte transporters, particularly those relevant to airway epithelial Cl- secretion, has occurred in the past few years. Application of microelectrode techniques to measure intracellular ion activities, transmembrane voltages, whole-cell currents, and single channels in membrane patches has greatly enhanced our ability to investigate conductive transport at the subcellular and now molecular level. These techniques coupled with experiments on ion substitution, transepithelial or transmonolayer electrolyte flux, and use of inhibitors specific for electrolyte transport pathways provide
ThE
3076
‘From port in presented
the Symposium Segmental Epithelial Electrolyte TransPulmonary Systems with Relevance to Cystic Fibrosis by The American Physiological Society at the 75th An-
nual Meeting of the Federation of American Societies for Experimental Biology, April 24, 1991, Atlanta, Georgia. 2Abbreviations: BCECF, 2’ ,7’-bis(2-carboxyethyl) 5(6)-carboxyfluorescein; CaCdPK, calcium, calmodulin-dependent protein kinase; CF, cystic fibrosis; DAG, diacylglycerol; DIDS, 4,4’-diisothiocyanostilbene 2,2’-disulfonic acid; 1P2, inositol bisphosphate; 1P3, inositol trisphosphate; I,, short circuit current; PD, potential
difference; pH1, intracellular pH; PIP2, phosphatidyl-4,5-bisphosphate; transmonolayer CFTR, cystic
resistance; IBMX, fibrosis transmembrane
PIP, phosphatidyl-4-phosphate; PLC, phospholipase C; R,, 3-isobutyl-1-methylxanthine; conductance regulator.
0892-6638/92/0006-3076/$01.50.
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TABLE
1. Electrolyte transporters mediating Na
Mechanism
of
or C1
movement
in airway
epithelium Subcellular
Airway
transport
segment
location
Electrogenic
Cl
K
Species
transport
Reference
mechanisms
channel: cAMP-regulated
Trachea
cAMP-regulated cAMP-regulated
Trachea, Trachea
nasal polyps
Ca2-regulated
Trachea,
nasal
Volume-regulated ? ?
Trachea, nasal epithehium Trachea, nasal epithelium Bronchiolar Clara cell Trachea Trachea Trachea Alveoli,
channel
Na
channel
epithehium
Apical
Swine
6
Apical Apical
Human Dog
?
Human
Apical ? ? Basolateral
Human Human Rabbit Human
15, 16, 46, 47 15 4 1 4 24 52
Basolateral Basolateral
Human Dog Rat
16 11, 13 58
Apical Apical ? ? Apical Apical
Human Dog Sheep Rabbit Rabbit Rat
50 15 23 24 35 29
type II
Trachea Trachea Bronchiole Bronchiolar Clara Alveoli, type II
cell
Electroneutral
Cl-/HCO3Na/H
exchange
exchange
Na’/HC03 NaCI(K)
cotransport cotransport
type II
?
Rat
40
Alveoli,
type II
?
Rat
40
Alveoli,
type II
?
Rat
37
? Basolateral ?
Rabbit
19, 20
Human
17
Basolateral
Human
18
Basolateral Basolateral
Dog Horse
17 22
Trachea Trachea Trachea,
nasal epithelium
epithelium
Trachea Trachea
information on the mechanism of electrolyte transport that carries the current across the cell monolayer and on the contribution of electroneutral transport pathways to transepithelial flux. As with large airway epithehial cells, modulation of transport pathways by hormones, second messengers, and mediators also provide vital information for characterization and differentiation of electrolyte transporters. The model for airway epithelial Cl secretion depicted in Fig. 1 is apparently consistent with results from studies of electrolyte transport in some mammalian trachea. However, new information on the presence of transporters other than channels and ATPases or of multiple types of transporters and their localization in different segments of the lung will lead to refinement of this model. This review is necessarily brief and, hence, only summarizes current findings on electrolyte transporters and their regulation.
Electrogenic
AIRWAY
EPITHELIUM
pathways
Cl channels Numerous C1 channel dependent C, or (CaCdPK)2
mechanisms
Alveoli,
Nasal
LARGE
transport
studies characterize airway Cl- conductance as a that is activated by 1) phosphorylation by cAMP protein kinase A, Ca2-dependent protein kinase Ca2-calmoduhin-dependent protein kinase or 2) alterations in osmolarity. Indeed, the van-
ety of stimulatory
agents as well as the ability of Ca2 ionoCl secretion in cystic fibrosis (CF) airway epithehial cells that are unresponsive to j3-adrenergic agents (1) suggests multiple populations of Cl channels, each responsive to a separate modulatory pathway. Support for this hypothesis comes from several studies. Canine tracheal epithehial cells maintained in culture and volume expanded in the presence of cAMP have four detectable anion channel types that range from 10 pS to 250 pS in conductance and differ further by anion-cation selectivity and by voltagedependent kinetic properties (2). Cyclic AMP increases channel activity in these cells (2, 3). Diverse C1 channels have also been observed in human airway epithelial cells. phores
to evoke
Reducing
medium
osmolarity
increases
channel
activity
in a
DIDS (4,41diisothiocyanostilbene-2,22disulfonic acid)sensitive manner (4). DIDS blocks C channels in a variety of cell types. The Ca2 ionophore A23187 induces a significant DIDS-sensitive current that varies with respect to time-dependent kinetics and peak voltage-current relationship (4, 5). CaCdPK inhibitory peptide blocks this response but not swelling-induced responses (4). The halide permeability of Ca2and cAMP-induced Cl- currents varied considerably (4, 5). Taken together, these findings indicate that Ca2 and cAMP activate different Cl channels in the apical membrane of human airway. Indeed, multiple anion channels may exist in this In swine trachea,
isoproterenol
membrane. Isc responses
that indicate
Cl- secretion
to
acetylchohine
differ
in several
and
ways
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APICAL
BASOLATERAL
airway epithelium point to dissimilar transitions from fetal to adult state. This raises questions about the number and responsiveness of apical Cl- channels to second messengers and as to the mechanisms that sustain a gradient favorable for Cl- exit during Cl secretion. K channels
CI Secretion in Airway Epithilium Figure
1. Model for Cl- driven secretion in airway epithelium. During fluid secretion, three transporters are active. Uptake of C1 is coupled to Na through a NaCI cotransporter in the basolateral membrane that may also transport membrane through a channel(s) ment of C1. The movement of C pled to the efflux of K through a brane. An Na,K-ATPase pumps Movement of Na through tight maintains electroneutrality.
K. C1 exits the cell at the apical allowing electrodiffusive moveout of the cell is electrically couchannel in the basolateral memNa Out of the cell into the serosa. junctions
from
serosa
to lumen
(6). First, from proximal to distal trachea, smaller responses to agonists are observed toward the distal trachea, with isoproterenol-induced currents showing a steeper gradient than acetyichohine. Second, the magnitude of the responses to both agonists in a single tissue sample are not correlated. Last, acetylcholine-induced secretion is independent of isoproterenol-induced secretion. These findings support a model for stimulation of parallel and independent secretory pathways, one adrenergic and the other cholinergic, in different cell types. Recent observations support the existence of basolateral Cl- channels. A reduction in serosal Cl- from 120 to 3 mM depolarizes the electrical potential across the basolateral membrane (7). This finding, together with an increased transepithelial resistance and decreased fractional apical resistance, indicates the operation of a Cl- conductance. Although this channel is considered minor, its function and regulation by hormones and mediators remain unknown. Developmental influences on Cl channel function appear in several animal species. In fetal sheep trachea, two studies showed that the predominant net transepithelial ion flow under both open circuit and short circuit conditions is C1 secretion (8, 9). However, the studies report different j3adrenergic C1 secretory responses. In one study (8), the C1 secretory response to /3-adrenergic agents in adult sheep is less than in fetal cells. In the other, Olver and Robinson (9) found substantial net Na flux from lumen to submucosa in fetal, newborn and adult sheep tissue. One interpretation of these findings is that /3-adrenergic secretory response seen in fetal trachea is progressively lost postnatally until reaching low adult levels. Rabbit trachea shares some of the developmental characteristics of sheep trachea. For example, fetal rabbit airway cells grown in in vitro culture display simultaneous Cl- secretion and Na4 absorption, with Na absorption in adult cells accounting for threefold more I than in fetal cells. In contrast to intact sheep trachea, epinephrine stimulates Cl secretion in both adult and fetal cells, with a higher response seen in adult cells (10). These few studies of the developmental aspects of electrolyte transport in large 3078
Vol. 6
September
1992
Cl and K conductances in apical and basolateral plasma membranes, respectively, are functionally linked and coordinated so that, during Cl- secretion, the conductances balance the rates of C1 and K4 flow across both membranes and stabilize cell volume (Fig. 1). In tracheal tissue permeabilized at the apical surface with amphotericin B, two different Ba-sensitive K conductances are distinguished by their sensitivity to quinidine (11). Cell swelling induces a third quinidine-sensitive K4 conductance and cell shrinkage blocks all three conductances. Each K4 channel also differs in its ion selectivity. Because hormone stimulation with epinephrine increases the quinidine-insensitive conductance, this K4 channel is thought to have a role in the Cl secretory response. At the subcellular level, two K4 conductances are distinguished by their sensitivity to charybdotoxin and by their role during a biphasic response to isoproterenol (12, 13). A charybdotoxin-sensitive K channel is responsible for the Ca2-dependent increase in the basolateral K4 permeability at the outset of cAMP-stimulated Cl- secretion. A second charybdotoxin-insensitive K channel is activated during the sustained component of Cl- secretion. Results from these and other studies indicate that Ca24-dependent K4 channel activation is responsible for increased K4 conductance during the Cl secretory response (14, 15) (Fig. 2). At the intracellular level, cr-adrenergic agents and intracellular Ca2, as well as cAMP-sensitive intracellular Ca2 stores, regulate K4 channel activity (15). Basolateral K channels in CF airway cells share similar con-
BASOLATERAL
APICAL
CI
-
Regulation of Cl - Secretion in Airway Epithelum Figure
2. Modulation
through
adrenergic
adrenergic receptors clase, respectively.
of airway receptors.
epithelial Basolateral
electrolyte a-adrenergic
transporters and
/3-
mediate activation of PLC and adenylate cyPLC produces a rapid breakdown of poly-
phosphoinositides and transient accumulation of IP3. IP3 mobilizes intracellular Car and DAG together with Ca2 activated protein kinase C. Elevated Ca2 alone opens the basolateral K4 channel whereas DAG plus Ca2-activated protein kinase C stimulate basolateral NaCl(K) cotransporter and apical C1 channel. Increased cAMP levels directly activate apical Cl- channels. Abbreviation: AR, adrenergic receptor.
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ductive and ion selectivity properties of channels in normal cells (14, 16). Regulation of these channels by Ca in excised, inside-out patches was also normal, which implies (as do results from studies of NaC1(K) cotransport) (17) that electrolyte transporters in the basolateral plasma membrane may be unaffected by the CF defect. Na channels In Na4-absorbing epithelium, including many mammalian airway epithelia, Na4 entry into the cell occurs through an amioride-sensitive Na4 channel in the apical plasma membrane (Fig. 3). The activity of this channel is closely related to the Na4 conductance of airway tissue (18). However, ascertaining the mechanism (or mechanisms) of regulation of airway epithelial Na4 conductance has proved to be difficult. Investigations on the developmental aspects of Na4 channel function show that fetal tracheal epithelial cells absorb less Na4 and secrete more C1 than adult cells (8) and that hormone stimulation of Cl- secretion differs markedly between fetal and adult cells (10). Expression of Na4 channels in genetic disease may be abnormal, as in it is CF, where excessive Na4 absorption by airway epithehium indicates a dysfunction in addition to defective Cl- secretion that has yet to be defined. Electroneutral
pathways
Two basic types of electroneutral electrolyte transporterscoupled transport of ions or exchange of ions - operate in airway epithelium. Both electrolyte transport mechanisms mediate ion transport with no net charge transfer. Early models of Cl- secretion depict uptake of Na and Cl- by coupled transport or cotransport with K4 in the basolateral plasma membrane (Fig. 1). Electrophysiologic studies demonstrate inhibition of spontaneous or induced C1 secretion with basolateral application of loop diuretics, such as bumetanide or furosemide, which are particularly effective in blocking renal NaK2C1-cotransport (15). More recent studies provide new information on the activity of a NaCl(K) cotransporter and its regulation by hormones, Ca24, and osmolarity (17, 19, 20). Most mammalian airway epithelia,
APICAL
BASOLATERAL
Na1’
Na Absorption in Airway Epithelium Figure 3. Model amiloride-sensitive
of Na Na
absorption in channel mediates
airway epithelium. Na entry at the
An apical
membrane. Na’ exits the cell through a Na,K-ATPase. Cl can move across the epithelium either through cells or through a paracellular pathway between cells.
AIRWAY TRANSPORTERS
with the exception of canine and bovine trachea, spontaneously absorb Na4. In human nasal epithelium and tracheal epithehium of human and rabbit, an NaCI(K) cotransporter is quiescent until stimulated by the endogeneous hormone epinephrine, by a-adrenergic agents, by Ca2 ionophore, or by hyperosmotic medium. Responses to hormones are blocked by a-adrenergic antagonists: a ,-adrenergic in human and a2-adrenergic in rabbits. Indeed, the a1-and a2adrenergic agonists methoxamine and clonidine, respectively, also stimulate loop diuretic-sensitive NaCl cotransport in human and rabbit airway epithelial cells, respectively (17, 19). Two lines of evidence are compatible with a-adrenergic activation of the cotransporter: 1) The failure of /3adrenergic agonist to activate bumetanide-sensitive Na4 and C1 transport (17, 19) and 2) inability of propranolol to block epinephrine-induced responses (19). Acute elevation of intracellular calcium with a calcium ionophore activates NaCI cotransport, suggesting a Ca2’-dependent mechanisms of signal transduction (Fig. 2). This was explored in cell cultures of isolated human or rabbit airway epithelial cells. In both cases, a-adrenergic agonists induce an increased breakdown of phosphatidyhinositol-4,5bisphosphate (PIP2) and transient accumulation of inositol-1,4,5-trisphosphate (1P3), which is compatible with activation of phospholipase C (PLC) (21). Pertussis toxin blocks the response in human cells. The kinetics of inositol lipid and inositol phosphate metabolism in normal human and CF airway cells differ only slightly, probably due to different tissue sources of epithehial cells, but call attention to a pattern of metabolism of phosphatidylinositol4-phosphate (PIP) and inositol-4,5-bisphosphate (IP2) that may involve direct action of phosphohipase C on PIP (21). Because CF cells resemble normal cells in these responses, we conclude that the CF defect does not perturb a- adrenergic receptormediated events in human airway cells, and indeed basolateral electrolyte transporters and basolateral enzymes essential for generation of intracellular second messengers are also unaffected. Thus, the CF defect may be limited to apical plasma membrane proteins. In rabbit cells, the a2-adrenergic agent clonidine activates NaCl(K) cotransport but fails to block /3-adrenergicstimulated adenylate cyclase activity. Thus, the activation of PLC (which liberates IP3, an agent that increases intracellular Ca24 levels by mobilizing nonmitochondrial pools of C a24) by clonidine may represent a mechanism of signal transduction unique to rabbit tracheal epithehial cells or to this receptor. As with cotransporter activation, the a2adrenergic antagonist blocks the changes in inositol lipid and inositol phosphate levels induced by epinephrine and clonidine. Hormones do not affect loop diuretic-sensitive K uptake in rabbit cells, suggesting a model of K4-independent NaCl coupled transport (20). Additional lines of evidence for this model include: 1) the stoichiometry of bumetanide-sensitive Na’ and Cl- uptake ranges from 1.0 with hyperosmotic perturbation to 1.4 with hormonal stimulation and 2) ion substitution of N-methylglucamine for Na4 or gluconate for C1 blocks hormone-stimulated uptake of the cotransported ion (20). The first evidence for the operation of electrolyte exchangers in airway epithehial ion transport comes from studies of equine trachea (22). Based on the sensitivity of Cl fluxes to basolateral bicarbonate and basolateral amiloride, models of Cl- secretion depict two dissimilar components: a bicarbonate-dependent Cltransport and bumetanidesensitive Cl- transport (Fig. 4). C1/HC03 exchange acts in
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depending on the species and environmental conditions. In rabbit bronchioles, nonciliated cells comprise approximately 70% of total epithelial cells. Clara cells grown in in vitro cell culture on collagen matrix supports display a PD of 31 mV and resistance of 500 ohm #{149} 2 (24). The much higher PD in cultured Clara cells vs. intact bronchioles may reflect species differences or differences in experimental systems. Electrophysiologic studies and radioisotopic flux measurements on cell monolayers indicate active Na’ absorption via an amioride-sensitive pathway on the apical surface. Active C1 secretion under baseline conditions is minimal; however, a significant portion of C1 flux in amiloride-treated monolayers occurs by as-yet-unidentified nonconductive pathways.
SEROSAL
model for electrolyte transport across equine C1 enters the cell at the basolateral plasma membrane through either NaCI(K) cotransport or C1/HC03 exchange, which is indirectly coupled to Na’/H4 exchange. Na4 enters the cell down its electrochemical gradient through the exchange pathway or amiloride-sensitive Na’ channels in the mucosal (apical)
Figure
tracheal
4. Tentative
plasma membrane. Cl efflux presumably occurs through C1 channels in the mucosal (apical) plasma membrane. Na4 is pumped from the cell by basolateral Na’,K-ATPase. K’ exits the cell through basolateral (serosal) Ba-sensitive K4 channels. Reproduced from ref 22 with permission.
parallel to Na4/H4 exchange to serve as a major influx pathway for Cl- and supports most of the basal C secretion observed in intact tissue. cAMP does not affect HCO3-dependent Cl- secretion but can induce a C1 secretion that is sensitive to basolateral application of bumetanide. We have yet to discern whether the C1/HC03 exchange or HC03-dependent Cl- secretion is regulated by different second-messenger system (or systems).
BRONCHIOLAR
EPITHELIUM
Bronchioles link the large airways and alveoli and are characterized by a small diameter and absence of cartilage and submucosal glands. This plus the cumulative large surface area represented by bronchioles suggest that bronchiolar epithelial cells are a potential site for the vectorial movement of electrolytes and water. Recently Al-Bazzaz et al. (23) reported the first direct measurement of ion transport in intact bronchioles by microperfusing bronchioles dissected from sheep lung. The spontaneous PD of 2.5 mV, lumen negative, is reduced by KCN. Ion substitution studies show that K4 and HCO3do not affect PD whereas Na’ and C1 conductance pathways operate spontaneously to mediate transepithelial ion movements. Treatment of tissue with epinephrine or the /3-adrenergic agonist isoproterenol increases the PD in a bumetanide-sensitive manner. This new information suggests that terminal bronchioles secrete C1 and water. The actual role of Na4 conductance pathways remains unknown. Nevertheless, microperfusion of intact bronchioles promises to provide new information on spontaneous ion transport mechanisms and their regulation by hormones. Bronchiolar
Clara
cells
Of the two cell types that comprise the bronchial nonciliated (Clara) cells are well characterized cally. Their distribution and relative numbers
3080
ALVEOLAR
EPITHELIUM
epithelium.
Vol. 6
September
1992
epithelium, morphologivary widely
Considerations of lung fluid transport invariably turn to alveolar epithelial cells and their role in electrolyte transport in fetal and postnatal lung. Fetal lung development proceeds sequentially through five stages, starting with an embryonic stage to the final alveolar stage. Because alveoli appear during only the terminal stages of lung development, only limited information can be obtained about the development or expression of alveolar electrolyte transport mechanisms. Nevertheless, experimental models ranging from in vivo lung micropuncture to whole-cell current recording from cultured alveolar cells provide insight on salt and water movement in the alveolus and on specific ion transporters that operate in this epithelium. Electrolyte
transport
in alveolar
tissue
Several lines of evidence support a critical role for fetal alveolar cells in lung fluid transport. First, interepithelial tight junctions connect fetal alveolar epithelial cells. Second, the alveolar epithelium restricts the transepithelial movement of macromolecules and small nonionic molecules. Overall vectorial flow of ions and water differs markedly in fetal and postnatal alveoli. In the former, active Cl- secretion promotes the flow of water into the fetal lung. In the latter case, a physiological signal triggers conversion to an Na4-absorbing epithelium. What constitutes this signal remains unknown. In both fetal and adult lung, cAMP induces fluid absorption. Perfusion of fetal lung with agonists that increase cAMP or with CAMP blocks fluid secretion and induces fluid absorption. In the adult rat lung, perfusion of alveolar spaces with a cAMP analog or phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) increases the paracellular permeability and the rate of fluid absorption and movement of 22Na from alveolar spaces to vascular spaces (25). The same agents stimulate an amioride-sensitive Na4 uptake from alveolar spaces to vascular spaces (26). These findings fit a model depicting Na4 absorption mediated through the uptake of Na4 by channels in the apical membrane (Fig. 3). Ion
transport
mechanisms
in alveolar
type
II cells.
With the advent of cell isolation techniques and cell culture methods, investigators have more systematically characterized electrolyte transport properties of alveolar type II cells grown as resistive monolayers in in vitro cell culture. One of the first observations suggesting that monolayers transport electrolytes and water is the formation of domes or blisters
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(27-29).
Application of cAMP analogs, phosphodiesterase and /3-adrenergic agents increases dome formation (28). These results along with finding that propranolol blocks the 3-adrenergic effects (28) suggest that net active transport of Na from medium with an obligatory osmotic flow of water to substratum causes dome formation and that increased intracellular cAMP levels stimulate epithelial Na4 transport. More direct evidence for this hypothesis and identification and characterization of specific electrolyte transporters came from studies with monolayers of type II cells grown on a membranous support. As seen in Table 2, the spontaneous PD, I,, and R1 values are quite similar to those observed in vivo between alveolar lumen and plural surface. In most experimental systems, monolayer resistance ranges from 200 to 600 ohm . cm2. Cells grown on tissue culture-treated Nucleopore filter supports develop resistances greater than 2000 ohm. cm2, suggesting the influence of the substratum on monolayer bioelectric properties (30). Indeed, Cott (31) cultured type II cells on either human amniotic basement membrane or collagencoated Milhipore filters and observed a higher resistance (491 vs. 291 ohm cm2) and lower short circuit current (2.85 vs. 4.51 iA/cm2) for the former substratum. Overall, type II alveolar cells cultured as monolayers retain the bioelectric properties of intact tissue. Fetal alveolar cells from 18, 19, and 21 day fetuses display a low PD but variable I,, and R (Table 2) (32, 33). The sensitivity of cyst formation and size by day 18 cells to bumetanide and cAMP indicates active Cl- secretion stimulated by cAMP (32). However, an amiloride-inhibitable component to ‘Sc has also been observed in fetal alveolar cells (33). Amiloride sensitivity increases with gestational age, indicating an greater dependence on Na4 as the currentcarrying ion. Correspondingly, Cl- dependent I,, decreases. inhibitors,
Whether
these
changes
accurately
reflect
the
transition
of a
C1-secreting epithelium to a Na4-absorbing epithelium will depend on critical evaluation of cell types isolated by different experimental maneuvers and of cell types that grow in in vitro cell culture. Na transport
mechanisms
Na channel Late-gestation ently absorb
fetal and Na4 through
mature alveolar type an amioride-sensitive
II cells apparNa4 channel
2. Bioelectric properties of rat alveolar type II epithelium
TABLE
Fetal R,0.
V,, mV
cm2
I,
sAJcm2
age, days
Reference
Alveolopleural -4.7
N.D.
59
N.D. Adult transepithelial
-0.7 -1.2
217 291
3.6 4.5
-9.7
2257
4.4
29 31
30
(30-33). Studies at the cellular and subcellular levels provide more information on the transport mechanism. At the cellular level, Na4 flux from apical to basolateral compartments predominate (30). /3-Adrenergic and cAMP-generating agents increase ‘Sc in an amiloride-sensitive manner that is not dependent on loop diuretics (29, 32, 34). This points to increased /3-adrenergic regulation of Na4 absorption. Support for this conclusion comes from ion substitution studies. Replacement of Na4 with choline significantly reduces I, in adult or fetal cells (30, 33). Substitution of Cl with gluconate has no effect on Membrane potential-dependent Na’ transport is also observed in membrane vesicles isolated from alveolar type II cells (35). These Na’ channels, because of their lower affinity to amioride and different pharmacological properties with respect to amiloride and its analog, are classed as a lowaffinity Na4 channel. The role of intracellular pH in inhibiting channel activity also differentiates the channel from high-affinity amiloride Na4 channels. Whether these properties indicate a location in a contaminating organelle, apical membrane, or basolateral plasma membrane has yet to be confirmed. Na-coupled transporters that are active in adult type II cells include Na4/H4 exchange (36, 38) and NaHCO3 cotransport (36). Using cells loaded with the pH-sensitive fluorescent probe 27-’bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), investigators probed Na’/H4 transport properties by acidifying cells with nigericin. Acidification is highly dependent on the Na4 content of extracellular medium. Amiloride at a concentration of 0.1 mM blocks recovery of pH (alkalinization). Substitution of Na4 for other cations shows that Li4, but not K4, Rb4, or Cs4, support cytoplasmic alkalinization (36). In cells with different preset pH1 values established in Na’-free medium, the rate of recovery of pH1 to Na4-replete medium depends on the initial pH1. Greater rates of alkalinization occur in cells in which pH1 is below pH 7.0 (36). At the plasma membrane level, Shaw et al. (39) demonstrated a saturable, amiloride-sensitive Na4 uptake that is independent of membrane potential. An butward facing H4 gradient supports Na4 uptake into apical membrane vesicles prepared from alveolar type II cells; hence, Na4 uptake is modeled as Na’/H4 exchange. The functional role of the Na4/H4 exchanger appears to be related to recovery of type II alveolar cells from an acid load but is probably not active at physiological pH. In cultured type II cell monolayers, NaHCO3 cotransport was detected in cells subjected to rapid acidification by CO2 pulse (37). pH1 recovery was reduced by substitution of choline for Na’ and by the stilbene DIDS. Replacement of Cl with gluconate and exposure to amiloride did not affect the rate of recovery. This Na4-dependent, Cl-independent transport mechanism is most consistent with NaHCO3 cotransport and may well function to regulate pH1 in alveolar epithelium, transport acid-base equivalents across the epithelium, and modulate pH of alveolar fluid. Localization of NaHCO3 cotransport and its stoichiometry have not yet been determined but are critical for modeling the operation of this transporter with other alveolar type II transporters to explain the movement of H4, HC03, and OH- across the epithelium.
Fetal transepit/aelial -2.3 -1.5 -4.2 -2.4
378 315 589 N.D. N.D
.,
not determined.
5.8 4.7 7.2 N.D.
18 19 21 18
33 33 33 32
Cl- transport
mechanisms
Nord et al. (40) identified a Na’-independent C1/HCO3 anion exchange pathway in confluent monolayers of rat type II alveolar cells. Alkalinization of BCECF-loaded cells by removal of HC03/C02 from the bathing medium induces a
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recovery with an absolute requirement for Cl-. The rate of recovery is inhibited by DIDS. Because the Na4/H exchange is inactive at physiological pH, Cl-/HCO3 exchange may be an more important determinant of steady-state pH1. Confirmation of this hypothesis requires more information on the localization of the Cl/HCO3-exchanger and buffering capacity of the cells at steady state.
ELECTROLYTE
TRANSPORT
IN CYSTIC
FIBROSIS
Cystic fibrosis (CF) is an autosomal recessive genetic disorder characterized by defective fluid secretion in epithelia of the sweat glands, small intestine, pancreas, and airways. Despite identification of a gene for CF (41, 42), we still do not how the defect manifests itself in each of the affected organs. Recent studies point to a role for the gene product as a Cl- channel in airway epithelial cells (43, 44). Because the CF defect compromises electrolyte absorption and secretion, research has focused on how the CF defect affects specific electrolyte transport mechanisms that operate during absorption or secretion. Defective
electrolyte
transport
in CF airways
Experimental systems ranging from in vivo measurement of potential difference to whole-cell patch clamp measurements provide compelling evidence for impaired Cl permeability and increased Na4 absorption in nasal, tracheal, and bronchial epithelia. Whether the CF defect also affects bronchiolar and alveolar epithelial electrolyte transport remains unknown. The basic abnormality in Cl permeability in CF airways becomes evident with the failure to stimulate Cl secretion by protein kinase C and protein kinase A although Cl channels in the apical plasma membrane have conductive and kinetic properties of normal Cl- channels. Elevated Ca2 still stimulates transepithelial Cl- secretion although the exact mechanism is not understood (7, 45). Identification of the defective Cl- channel first focused on rectifying Cl channels of about 40 pS in the apical plasma membrane of tracheal epithelial cells (16, 46). Subsequent studies of the epithelium of the CF nasal turbinate provide evidence for a 20 pS nonrectifying Cl- channel as the most common C1 channel in normal human airway cells (47). Phosphorylation by protein kinase A activates the 20 pS Cl channel. In CF cells, open probability of this channel is reduced as is the number of 20 pS Cl- channels compared with other Cl- channels. Other studies suggest the presence of three different Cl channels in CF airway epithelial cells each characterized by different conductive and kinetic properties as well as activation by specific stimuli (4, 5). In CF cells, Car-activated Cl channels differ little from normal cells. The opposite occurs with stimuli that increase intracellular cAMP levels and volume regulate upon exposure to hypoosmotic medium. While current hypotheses on the CF defect associate the error in airway Cl- permeability with a complex mechanism of regulation, more information must be obtained on the contribution of different apical Cl channels to Cl secretion and their regulation. Observations using incised normal and CF nasal tissue and primary cultures of isolated airway epithelial cells demonstrate a high (>90%) amiloride-sensitive ‘sc and Na4 transepithelial flux that are compatible with Na4 absorption (1, 48). Microelectrode measurement of electric potentials demonstrate an ‘Sc approximately twofold higher than normal in CF airway cells but with normal intracellular Na4 ac-
3082
Vol. 6
September
1992
tivity (18, 49, 50). Reports of an amioride-insensitive Na4 channel in normal and CF cells raise the possibility of a role for this channel in excessive Na4 absorption in CF (50, 51). Overall,
a model
of greater
Na’
channel
activity
in the
apical
plasma membrane of CF airway cells emerges. The increased Na4 absorption coupled to a relative Cl- impermeability that prevents secretion of ions and fluids may play a major role in the high viscosity of airway mucus. Unaffected
electrolyte
transporters
in CF airway
The defect in CF appears focused to fluid secretion stimulated by /3-adrenergic agonists or agents that increase CAMP levels by bypassing receptors. Other epithelial transporters that function during absorption or secretion share conductive and/or kinetic properties of normal transporters and similar regulatory mechanisms. All indications thus far point to normal activation of unaffected transporters that include NaC1(K) cotransport (17), basolateral K’ channels (16), and basolateral Cl- channel (52). Cystic
fibrosis
transmembrane
conductance
regulator
Isolation of the gene for CF opened new opportunities to explore the molecular basis of the disease. The gene encodes a protein of 1,480 amino acids that is called the cystic fibrosis transmembrane conductance regulator (CFTR). The predicted protein resembles multidrug resistance proteins and has two repeats consisting of an ATP-binding domain followed by six transmembrane domains. Connecting the two halves of CFTR is a regulatory domain that has multiple sites for phosphorylation by protein kinases (41). Of the different mutations of CFTR gene, the most common CF mutation, a deletion of three nucleotides that encode phenylalanine 508 (SF508), is present on 70% of all CF chromosomes. Antibodies to a synthetic peptide corresponding to position 505-511 and including phenyalanine 508 blocks a cAMP-dependent Cl- current activation and attenuates volume activated Clcurrents but leaves Ca2-dependent anion currents unaffected (53). These findings complement studies (43, 44) suggesting a role for CFTR in anion transport in epithelial cells and further suggest an involvement with other anion transporters. Introduction of full-length CFTR cDNA to nonepithelial cell lines induces expression of cAMP-dependent C1 conductance (44, 54). An Sf9 insect cell line infected with a baculovirus expression vector system and mouse fibroblast cell lines transfected with plasmid vector pCOF-1 acquire the ability to synthesize CFTR with localization in the cell surface and intracellular membranes. The inducible cAMPdependent conductance closely resembles the linear currentvoltage relationship of CFTR expressed in epithelial cells (55). These models will prove advantageous in evaluating the structure-function relationship and regulation of CFTR. Numerous mutations in CFTR point to a lack of correlation between severity of CF lung disease and F508 mutation (56). Support for this conclusion comes from a recent report that patients with nonsense mutations in each CF gene lack CFTR mRNA in airway epithelial cells but have mild pulmonary disease and severe pancreatic disease (57). Clearly, the role of CFTR in the expression of CF needs clarification. Information from these studies points to several lines of investigation to ascertain whether CFTR is a regulated Cl- channel, a general transporter, or a protein that regulates other membrane or cytosolic proteins that make up the channel.
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CONSIDERATIONS
FOR
FUTURE
cotransport L125-L129
DIRECTIONS
New information on electrolyte transporters functioning in pulmonary segments will lead to refinement of models for Na’ absorption and Cl- secretion that reflect the diversity of the transport mechanisms. Future investigations that explore intracellular mechanisms of signal transduction linked to regulation of transporters by osmolarity, hormones, mediators, and pH will allow further differentiation of similar pathways operating in the same or different pulmonary segment. This information together with studies of developmental changes in transporters are needed to develop pharmaceutical approaches to circumvent defective transporters such as those expressed in CF. We know little of the structure of airway electrolyte transporters and how this structure and resultant function are affected by alterations in membrane lipid and polypeptide composition.
18.
21.
22.
23. 24.
1. Willumsen, N. J., and Boucher, R. C. (1989) Activation of an apical Cl conductance by Ca24 ionophores in cystic fibrosis airway epithelia. Am. j Physiol. 256, C226-C233 2. Shoemaker, R. L., Fnzzell, R. A., Dwyer, T M., and Farley,J. M. (1986) Single chloride channel currents from canine tracheal epithelial cells. Biochim. Biophys. Acta 858, 235-242 3. Schoppa, N., Shorofsky, S. R., Jow, F., and Nelson, D. J. (1989) Voltage-gated chloride currents in cultured canine tracheal epithelial cells. j Membr. Biol. 108, 73-90 4. Chan, H. C., Goldstein, J., and Nelson, D. J. (1992) Alternate pathways for chloride conductance activation in normal and cystic fibrosis airway epithelial cells. Am. j Physiol. In press 5. Anderson, M. P., and Welsh, M. J. (1991) Calcium and cAMP activate different chloride channels in the apical membrane of normal and cystic fibrosis epithelia. Proc. NatI. Acad. &i. USA 88, 6003-6006 6. Farley, J. M., Adderholt, G., and Dwyer, T. M. (1991) Autonomic stimulation of short circuit current in swine trachea. Life Sci. 48, 873-880 7. Willumsen, N. J., Davis, C. W., and Boucher, R. C. (1989) Intracellular Cl activity and cellular Cl- pathways in cultured human airway epithelium. Am. j Physiol. 256, C1033-C1044 8. Cotton, C. U., Lawson, E. E., Boucher, R. C., and Gatzy, J. T. (1983) Bioelectric properties and ion transport of airways excised from adult and fetal sheep. j Appi. Physiol. 55, 1542-1549 9. Olver, R. E., and Robinson, E. J. (1986) Sodium and chloride transport by the tracheal epithelium of fetal, new-born and adult sheep. j Physiol. (London) 375, 377-390 10. Zeitlin, P. L., Loughlin, G. M., and Guggino, W. B. (1988) Ion transport in cultured fetal and adult rabbit tracheal epithelia. Am. j Physiol. 254, C691-698 11. Butt, A. G., Clapp, W. L., and Frizzell, R. A. (1990) Potassium conductances in tracheal epithelium activated by secretion and cell swelling. Am. j Physiol. 258, C630-C638 12. McCann, J. D., Matsuda, J., Garcia, M., Kaczorowski, G., and Welsh, M. J. (1990) Basolateral K’ channels in airway epithelia. I. Regulation by Ca’ and block by charybdotoxin. Am. j PhysioL 258, L334-L342 13. McCann, J. D., and Welsh, M. J. (1990) Basolateral K’ channels in airway epithelia. II. Role in Cl- secretion and evidence for two types of K channel. Am. j Physiol. 258, L343-L348 14. Kunzelmann, K., Pavenstadt, H., and Greger, R. (1989) Characterization of potassium channels in respiratory cells. II. Inhibitors and regulation. Pftuegers Arch. 414, 297-303 15. Welsh, M. J. (1987) Electrolyte transport by airway epithelia. Physiol. Rev. 67, 1143-1184 16. Welsh, M. J., and Liedtke, C. M. (1986) Chloride and potassium channels in cystic fibrosis airway epithelium. Nature (London) 322, 467-470 17. Liedtke, C. M. (1989) a-Adrenergic regulation of Na-Cl
epithelia.
Am.
J. PhysioL 257,
N. J., and Boucher, R. C. (1991) Sodium transport and intracellular sodium activity in cultured human nasal epithelium. Am. j Physiol. 261, C319-C331 C. M.
(1990)
Calcium
and
a-adrenergic
regulation
of
Na-Cl(K) cotransport in rabbit tracheal epithelial cells. Am. J. Physiol. 259, L66-L72 Liedtke, C. M. (1992) Bumetanide-sensitive Na and Cl uptake in rabbit tracheal epithelial cells is stimulated by neurohormones and by hypertonicity. Am. j Physiol. In press. Liedtke, C. M. (1992) a1-Adrenergic signaling in human airway epithelial cells involves inositol lipid and phosphate metabolism. Am. j Physiol. In press Tessier, G. J., Traynor, T R., Kannan, M. S., and (YGrady, S. M. (1990) Mechanisms of sodium and chloride transport across equine tracheal epithelium. Am. j PhysioL 259, L459-L467 A1-Bazzaz, F. J., Tarka, C., and Farah, M. (1991) Microperfusion of sheep bronchioles. Am. j Physiol. 260, L594-L602 Van Scott, M. R., Davis, C. W., and Boucher, R. C. (1989) Na’ and
REFERENCES
airway
Willumsen,
19. Liedtke,
20.
in human
C1
transport
across
rabbit
nonciliated
bronchiolar
(Clara) cells. Am. J. Physiol. 256, C893-C901 G., Basset, G., Bouchonnet, F., and Crone, C. (1987) cAMP and /3-adrenergic stimulation of rat alveolar epithelium: effects on fluid absorption and paracellular permeability. Pftuegers Arch. 410, 464-470 26. Goodman, B. E., Anderson, J. L., and Clemens, J. W. (1989) Evidence for regulation of sodium transport from airspace to vascular space by CAMP. Am. j Physiol. 257, L86-L93 epithelial 25. Saumon,
27. Goodman,
Evidence
B. E., Fleischer,
for active
pulmonary
alveolar
R.
S., and
Na’ transport epithelial
Crandall,
by cultured cells. Am. j
E. D. (1983)
monolayers of Physiol. 245,
C78-C83 28. Goodman, Regulation 29.
30.
31.
32.
33.
34.
B. E., Brown, S. E. S., and Crandall, E. D. (1984) of transport across pulmonary alveolar epithelial cell monolayers. j AppI. Physiol. 57, 703-710 Mason, R. J., Williams, M. C., Widdicombe, J. H., Sanders, M. J., Misfeldt, D. S., and Berry, L. C., Jr. (1982) Transepithelial transport by pulmonary alveolar type II cells in primary culture. Proc. Nail. Acad. 54. USA 79, 6033-6037 Cheek, J. M., Kim, K. J., and Crandall, E. D. (1989) Tight monolayers of rat alveolar epithelial cells: bioelectric properties and active sodium transport. Am. J. Physiol. 256, C688-C693 Cott, G. R. (1989) Modulation of bioelectric properties across alveolar type II cells by substratum. Am. J. Physiol. 257, C678-C688 McCray, P. B., Jr., and Welsh, M. J. (1991) Developing fetal alveolar epithelial cells secrete fluid in primary culture. Am. J. Physiol. 260, L494-L500 Rao, A. K., and Cott, G. R. (1991) Ontogeny of ion transport across fetal pulmonary epithelial cells in monolayer culture. Am. j Physiol. 261, L178-L187 Cott, G. R., Sugahara, K., and Mason, R.J. (1986) Stimulation of net active ion transport across alveolar monolayers. Am. j Physiol. 250, C222-227
type
II
cell
35. Matalon, S., Bridges, R. J., and Benos, D. J. (1991) Amiorideinhibitable Na’ conductive pathways in alveolar type II pneumocytes. Am. j PhysioL 260, L90-L96 36. Nord, E. P., Brown, S. B. S., and Crandall, E. D. (1987) Characterization of Na’-H antiport in type II alveolar epithelial cells. Am. j PhysioL 252, C490-C498 37. Lubman, R. L., and Crandall, E. D. (1991) Na-HC03 symport
modulates
intracellular
pH
in alveolar
epithelial
cells.
Am.
j
PhysioL 260, L555-L561 38. Sano, K., Cott, G. R., Voelker, D. R., and Mason, R. J. (1988) The Na’/H antiporter in rat alveolar type II cells and its role in stimulated surfactant secretion. Biochim. Biophys. Acta 939, 449-458 39. Shaw, A. M., Steele, L. W., Butcher, P. A., Ward, M. R., and Olver, R. E. (1990) Sodium-proton exchange across the apical membrane of the alveolar type II cell of the fetal sheep. Biochim. Biophys. Acta 1028, 9-13 40. Nord, E. P., Brown, S. E. S., and Crandall, E. D. (1988) Cl/HC03 exchange modulates intracellular pH in rat type II
AIRWAYTRANSPORTERS 3083 w.fasebj.org by Univ of So Dakota Lommen Hlth Sci Library (192.236.36.29) on December 21, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNum
alveolar epithelial cells. j BioL C/atm. 263, 5599-5606 41. Riordan, J. R., Rommens, J. M., Kerem, B., Alon, N., Rozmahel, R., Grzelczak, Z., Zielenski, J., Lok, S., Plavsic, N., Chou, J.-L., Drumm, M. L., lannuzzi, M. C., Collins, F. S., and Tsui, L.-C. (1989) Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245, 1066-1072 42. Kerem, B. S., Rommens, J. M., Buchanan, J. A., Markiewicz, D., Cox, T K., Chakravart, A., Buchwald, M., and Tsui, L. C. (1989)
43.
44.
45.
46.
47.
48.
49.
Identification
of the cystic
fibrosis
gene:
epithelia.
Abnormal
clase activation. 50. Willumsen, N.
j
J.,
basal
rate
and
response
analysis.
Vol. 6
to adenylate
Clin. Invest. 78, 1245-1252 and Boucher, R. C. (1991) Transcellular
dium transport in cultured cystic fibrosis epithelium. Am. j Physiol. 261, C332-C341
3084
genetic
Science 245, 1073-1080 Anderson, M. P., Gregory, R. J., Thompson, S., Souza, D. W., Paul, S., Mulligan, R. C., Smith, A. E., and Welsh, M. J. (1991) Demonstration that CFTR is a chloride channel by alteration of its anion selectivity. Science 253, 202-207 Rommens, J. M., Dho, S., Bear, C. E., Kartner, N., Kennedy, D., Riordan, J. R., Tsui, L. C., and Foskett, K. (1991) cAMPinducible chloride conductance in mouse fibroblast lines stably expressing the human cystic fibrosis transmembrane conductance regulator. Proc. NatL Acad. Sci. USA 88, 7500-7504 Clancy, J. P., McCann, J. D., Li, M., and Welsh, M. J. (1990) Calcium-dependent regulation of airway epithelial chloride channels. Am. J. Physiol. 258, L25-L32 Frizzell, R. A., Rechkemmer, G., and Shoemaker, R. L. (1986) Altered regulation of airway epithelial cell chloride channels in cystic fibrosis. Science 233, 558-560 Duszyk, M., French, A. S., and Man, S. F. P. (1989) Cystic fibrosis affects chloride and sodium channels in human airway epithelia. Can. J. Physic!. Pharroacol. 67, 1362-1365 Knowles, M., Gatzy, J., and Boucher, R. (1983) Relative ion permeability of normal and cystic fibrosis nasal epithelium. j Clin. Invest. 71, 1410-1417 Boucher, R. C., Stutts, M. J., Knowles, M. R., Cantley, L., and Gatzy, J. T. (1986) Na’ transport in cystic fibrosis respiratory
September
1992
human
cy-
sonasal
51. Jorissen, M., Vereecke, J., Carmeliet, E., Van den Berghe, H., and Cassiman, J. J. (1991) Non-selective cation and dysfunctional chloride channels in the apical membrane of nasal epithelial
cells cultured
from
cystic
fibrosis
patients.
Biochim.
Bio-
phys. Ada 1096, 52-59 52. Willumsen, N. J., Davis, C. W., and Boucher, R. C. (1989) Cellular Cl transport in cultured cystic fibrosis airway epithelium. Am. j PhysioL 256, C1045-C1053 53. Chan, H. C., Kaetzel, M. A., Nelson, D. J., Hazarika, P., and Dedman, J. R. (1991) Antibody against a CFTR-derived synthetic peptide inhibits anion currents in human colonic cell line T84. j BioL Chem. In press N., Hanrahan, J. W., Jensen, T. J., Naismith, A. L., Sun, S., Ackerley, C. A., Reyes, B. F., Tsui, L. C., Rommens, J. M., Bear, C. E., and Riordan, J. R. (1991) Expression of the
54. Kartner,
cystic
fibrosis
gene
in non-epithelial
invertebrate
cells produces
a regulated anion conductance. Cell 64, 681-691 55. Cliff, W. H., and Frizzell, R. A. (1990) Separate C1 conductances activated by CAMP and Ca2 in C1-secreting epithelial cells. Proc. Nail. Acad. &i. USA 87, 4956-4960 56. Kerem, B. S., Zielinski, J., Markiewicz, D., Bozon, D., Gazit, E., Yahav, J., Kennedy, D., Riordan, J. R., Collins, F S., and Rommens, J. M. (1990) Identification of mutations in regions corresponding to the two putative nucleotide (ATP) binding folds of the cystic fibrosis gene. Proc. Nail. Acad. Sci. USA 87, 8447-8451
57. Hamosh, Rafizadeh,
A., Trapnell, C., Rosenstein,
B. C., Zeitlin, P. L., MontroseB. J., Crystal, R. G., and Cutting,
G. R. (1991) Severe deficiency of cystic fibrosis transmembrane conductance regulator messenger RNA carrying nonsense mutations R553X and W1316X in respiratory epithelial cells of patients with cystic fibrosis. j Clin. Invest. 88, 1880-1885 58. DeCoursey, T. E.,Jacobs, E. R., and Silver, M. R. (1988) Potassium currents in rat type II alveolar epithelial cells. j Physiol. (London) 395, 487-505
59. Ballard, S. T, Gatzy, J. T. (1991) Alveolar transepithelial potential difference and ion transport in adult rat lung. j AppL PhysioL 70, 63-69
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