Regulation of intracellular pH in alveolar epithelial cells.

Regulation RICHARD

of intracellular L. LUBMAN

AND

pH in alveolar EDWARD

epithelial

cells

D. CRANDALL

Will Rogers Institute Pulmonary Research Center, Division of Pulmonary University of Southern California, Los Angeles, California 90033

and Critical

Care Medicine,

Lubman, Richard L., and Edward D. Crandall. Regulation of intracellular pH in alveolar epithelial cells. Am. J. PhysioZ. 262 (Lung Cell. 1MoL. PhysioZ. 6):Ll-L14, 1992.-Alveolar type II epithelial cells in adult mammalian lungs actively transport salt and water, secrete surfactant, and differentiate into type I cells under normal conditions and following lung injury. It has become increasingly apparent that, like all epithelial cells, alveolar pneumocytes have evolved specialized ion transport mechanisms by which they regulate their intracellular pH (pHi). pHi is an important biological parameter in all living cells whose regulation is necessary for normal cellular homeostasis. pHi, and the ion transport mechanisms by which it is regulated, may contribute to many cellular processes, including transcellular transport, cell volume and osmolarity regulation, and intracellular electrolyte composition. Moreover, changes in pHi may serve as intracellular signals for biological processes such as cell growth, proliferation, and differentiation. We review herein the general principles of pHi regulation in epithelia and describe the mechanisms and effects of pHi regulation in alveolar pneumocytes. Many of the critical issues in current pulmonary research involve processes that pHi is most likely to affect, including maintenance of alveolar epithelial barrier integrity, development and maintenance of epithelial polarity, epithelial proliferation and differentiation, and regulation of transepithelial transport with respect to alveolar fluid balance in normal individuals and in those with excess alveolar fluid (i.e., pulmonary edema). Investigations into the regulation of pHi in alveolar pneumocytes and the regulatory effects of pHi in turn on other cellular processes are likely to yield information important to the understanding of lung biology and pulmonary disease. alveolar epithelium; alveolar homeostasis; alveolar fluid pH EPITHELIAL CELLS are subject to an environment marked by rapid changes in CO2 tension (Pco~) due to the respiratory cycle and to hypo- or hyperventilation in vivo, making regulation of intracellular pH (pHi) especially important for homeostasis in these cells. To this end, alveolar pneumocytes have evolved mechanisms for acid-base balance that are compatible with cell survival and capable of responding rapidly to stimuli from the environment. In addition, the mechanisms and effects of pHi regulation in alveolar pneumocytes have been implicated to have many additional consequences for the function and biology of these cells. These include stimulation of surfactant secretion (Zl), transepithelial transport of acid/base equivalents (66), and modulation of the extracellular alveolar milieu (78). Considerable data have been compiled on these and other effects of pHi for both epithelial and nonepithelial cell types (10, 39), suggesting a broad role for pHi in cellular regulation and signal transduction. For example, pHi has been shown to affect macromolecular tra .nsport (30), ion transpo rt and intracellular electrolyte composition (69, 97), cell-cell junctions (58), cytoskeletal protein interactions (56, 114), and cell morphology, growth, and differentiation (8, 96). ALVEOLAR

1040-0605/92

$2.00

Copyright

DETERMINANTS

OF PHi:

GENERAL

CONSIDERATIONS

Intracellular ( [H+]i) and extracellular hydrogen ion concentrations ( [H+] 0) would be identical in eucaryotic cells were the cells metabolically inert and their plasma membranes unselectively permeant. Consistent with the fact that neither of these conditions exists in living cells, it has been repeatedly demonstrated that pHi may, and generally does, differ from extracellular pH (pH,) (10, 39, 68, 88). Moreover, pHi has been shown to be tightly controlled in eucaryotes, varying in predictable fashion under different metabolic and developmental states. Intracellular acid accumulates primarily as a by-product of metabolic processes. The central problem of pHi regulation has therefore been defined as the neutralization of intracellular acid (88). Because the plasma membrane is relatively permeant to small uncharged molecules and only very selectively permeant to large or charged species, purely passive fluxes of weak acids and bases may also contribute to the net accumulation or excretion of acid. CO2 freely enters the cell, for example, whereas its conjugate base HCO: can only enter or exit by specialized membrane transport mechanisms. Furthermore, the plasma membrane potential ( Vm), a con-

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sequence of the plasma membrane permeability properties and the effects of concentration gradients, is generally negative in the cytoplasm of most cells. This adds an additional electrical driving force for hydrogen ions (i.e., acid) to enter cells. The tendency for cells to accumulate acid (or occasionally base) is thus a fundamental property derived from metabolic necessity and biological design. The cell has several mechanisms for the short-term handling of acid or base. These include cellular consumption by alternate metabolic pathways, intracellular sequestration by transfer between cytoplasm and organelles, and physicochemical buffering. Of these, the buffering properties of the cytoplasm, mostly derived from intracellular bicarbonate and phosphate buffers and proteins, are probably most important in neutralizing rapid changes in hydrogen ion concentration. The intracellular buffer capacity (pi), which can be defined as the tendency to resist changes in pHi upon addition of intracellular acid or base, can be determined for individual cells and generally falls between 10 and 100 mmol acid (or base) per change in pH unit per liter of cell water (88). Because cells do not have an infinite capacity to consume, sequester, or buffer hydrogen ions, long-term or steady-state regulation of pHi cannot depend solely on the aforementioned mechanisms and requires the net extrusion of acid (or base) from the cell for its maintenance. As a result, all eucaryotic cells have evolved specialized transport mechanisms facilitating the movement of acid-base equivalents across the plasma membrane. These transport mechanisms all require cellular energy, either directly or indirectly. For example, cellular energy is directly expended when hydrolysis of ATP results in proton extrusion via a proton-translocating adenosinetriphosphatase (H+-ATPase). This is an example of primary active transport (103). Conversely, energy for the electroneutral exchange of sodium and hydrogen ions via the Na+-H+ antiporter is supplied solely by the electrochemical gradient for Na+ to enter the cell, making sodium entry and acid extrusion thermodynamically favorable. This is an example of secondary active transport (3). Epithelial barriers characteristically have luminal (apical) and serosal (basolateral) aspects functionally compartmentalized by cell-cell junctions (48). This polarity, which is the structural and functional hallmark of epithelia, makes vectorial transport across epithelial surfaces possible. In addition to the housekeeping function of pHi regulation, epithelial cells can effect transcellular transport of acid and base due to the asymmetric distribution of specialized membrane transport systems. Thus it is particularly apparent that the transport mechanisms in epithelial membranes, generally ion transport mechanisms, are capable of performing multiple overlapping functions. In addition to pHi regulation, these ion transport mechanisms may contribute to transepithelial transport of water and solutes, modulation of intracellular and extracellular electrolyte composition, and regulation of cell volume and osmolarity. Furthermore, pHi and the ion transport mechanisms by which it is regulated can be stimulated by multiple extracellular factors. These include soluble cell factors (i.e., hormones, mito-

REVIEW

gens, and cytokines) (41, 106) and cell-cell and cellsubstratum interactions (72, 96). The relationship between pHi regulation and some of these processes has been recently reviewed (75, 102). All of these features define the process and regulation of pHi in eucaryotes, and in epithelia in particular. Although the fundamental biophysical determinants of pHi are universal in all cells, the precise mechanisms by which pHi regulation is effected may vary among individual cell types, tissues and species, in addition to variations based on developmental or metabolic state. It is therefore necessary to establish, for any individual cell type, the specific mechanisms and effects of pHi regulation. METHODS

FOR

MEASURING

PHi

In recent years, several techniques have been used to measure pHi in living cells. Although they have been the subject of previous general reviews (10, 39, 68, Ss), we briefly describe these methods herein. As has frequently been the case in pulmonary research, the complex architecture and cellular heterogeneity of the lung can pose formidable technical problems for their application. A variety of different microelectrode techniques have been used to measure intracellular ion activities, pH, and the electrical potential across cell membranes. For measurements of pHi, a cell must be impaled by two microelectrodes or one double-barreled electrode, one pH sensitive and the other an indifferent reference. The voltage difference between the two electrodes is a linear function of pH (88). Microelectrodes are capable of precise and reproducible measurement of intracellular pH in individual cells with excellent time resolution (~0.1 s), as evidenced by numerous studies in renal (12, 100) and other (57) epithelial cells. Such studies are limited by several considerations. First, the manipulation of such microelectrodes, generally microns in diameter, requires considerable dexterity. Moreover, even allowing for sufficient skill, some cells are too small to accommodate an appropriate electrode. Second, estimates of pH may be distorted by technical factors such as sensitivity of glass electrodes to cations at high pH (88). Third, microelectrode techniques are not easily adaptable to cells situated within highly compliant tissues, as is the case for alveolar epithelial cells in situ. Finally, microelectrode measurements are confounded by imprecise localization of a specific impalement (e.g., cytoplasm vs. organelle). pHi has also been estimated by a method utilizing the distribution of weak acids or bases. These methods are based on the assumption that only the uncharged forms of these electrolytes are permeant into cells and that their intra- and extracellular concentrations are the same after sufficient length of exposure (88). The concentration of the conjugate acid-base pair is determined, and pHi can be calculated if the acid dissociation constant (p&J for the pair is known. In practice, the indicator is generally a radioactive tracer (e.g., labeled 5,5-dimethyloxazolidine,@dione or DMO), and the ratio of intrato extracellular radioactivity is determined. Aside from the many theoretical limitations of this technique (e.g., effects of compartmentation), a major disadvantage is


INVITED

that it requires destruction of the cells. Furthermore, since only a steady-state measurement of intracellular pH can be made, this method has largely been supplanted by the fluorescent indicator techniques described below. Over the past few years, the use of fluorescent pHsensitive indicators has made the measurement of pHi in a large variety of living cells possible. Most of the studies to date in which pHi was measured in alveolar epithelial cells have used these probes. Fluorescence measurements of pHi have the advantage of being reasonably precise with good time resolution, while being relatively simple and nondestructive. This technique has been adapted for use in tissues, cultured cells, cell suspensions, and single cells (60, 74, 80, 87), as well as for measuring the pH of several types of extracellular fluids (29). In addition, pHi measurements using fluorescent indicators have been made using standard fluorometric techniques and microspectrofluorometry as well as fluorescence-activated cell sorting (FACS) or flow cytometry (65, 115). A large number of such pH-sensitive dyes have been synthesized by Tsien and others (52,87), although 2’,7’bis(2-carboxyethyl)-5(6)-carboxy-fluorescein (BCECF) has been used with widest success. The synthesis of esterified forms of this compound (e.g., BCECF-acetoxymethyl ester), which are trapped intracellularly upon cleavage of the ester group by cytoplasmic enzymes, has improved their utility. When BCECF emission is measured at a wavelength of 530 nm, this fluorescein derivative is maximally fluorescent intracellularly when excited at 503 nm in alveolar pneumocytes (79). Fluorescence is pH sensitive over the physiological range of pH 6.0-8.0 at an excitation wavelength of 503 nm but insensitive to pH changes at an excitation wavelength of 440 nm, an isosbestic point. This property of the dye makes it possible to factor out variations in cell volume, uptake, and dye leakage by relating the ratio of fluorescence at these two excitation wavelengths to pHi. Several methods have been devised to calibrate fluorescence ratio with respect to pHi (36, 109). We have utilized the K+-H+ ionophore nigericin according to the method of Thomas et al. (109). BCECF-loaded alveolar pneumocytes are exposed to a high-K+ buffer (130 mM) in the presence of nigericin such that intracellular concentration of K+ ( [K+] i) equals extracellular concentration of K+ ([K+],). Because [H’]; must then also approximate [H+],, pHi is effectively “clamped” to the pH of the much larger extracellular fluid compartment. Calibration by this method has been used in a wide variety of cell types with excellent correlation to other methods of pHi measurement. At least one other method, nuclear magnetic resonance (NMR), has been employed to measure lung pH. NMR measurements utilize pH-sensitive shifts in the frequencies of 31P spectra after application of an intense magnetic field to measure [ H+]i (88). Technical aspects of such measurements aside, pHi can be measured rapidly, repeatedly, and nondestructively by NMR in living cells and tissues. A major limitation of this technique appears to be the need for a large enough amount of intracellular 31P (i.e., high cell density) for a signal to be detected, thus hindering its application to the measurement of pHi in isolated alveolar epithelial cells. For example, in using

L3

REVIEW

NMR to measure total lung pH (including a contribution from pHi), the investigators needed to degas the organ to increase the amount of tissue per unit volume and to eliminate multiple gas-tissue interfaces (84). Nonetheless, technical improvements in NMR may be expected to facilitate such measurements in the future. MECHANISMS IN ALVEOLAR

OF PHi REGULATION EPITHELIAL CELLS

A very large and diverse number of pH;-regulating membrane transport mechanisms, primarily ion transporters, have evolved in eucaryotic cells. Many, although not all, of these have been described in epithelial cells. As noted above, these processes share several important properties. They are regulated by intra- and extracellular ion activities and pH, they can be stimulated or inhibited by soluble cell factors and cell-cell interactions, and their expression and/or activity may vary as a function of metabolic or developmental state. The ion transport mechanisms effecting pHi regulation in epithelial cells either utilize cellular energy directly (primary active transport, e.g., H+-ATPase) or indirectly (secondary active transport, e.g., Na+-H+ antiport). These mechanisms can also be categorized by whether or not they require the co- or countertransport of another ion or ions. Finally, as a practical matter, it is often useful to discuss bicarbonate-dependent pH;-regulating ion transport mechanisms separately from others. Na+-H+ antiport. The Na+-H’ antiporter (or exchanger), a ubiquitous ion transport mechanism involved in pHi regulation, was the first such mechanism to be described in alveolar pneumocytes. Initially described in the rabbit kidney and small intestine (76), it has since been found in essentially all epithelia. It contributes to many overlapping cell functions, including regulation of pHi and ion activities, regulation of cell volume and osmolarity, and transepithelial transport of acid/base equivalents, salt, and water. This bicarbonate-independent transport mechanism effects the one-for-one exchange of Na+ (or Li+) for H+ in an electroneutral fashion (that is, without the net transfer of charge across the cell membrane). The antiporter is driven by the preexisting electrochemical gradient for Na+ to enter the cell and, although both theoretically and experimentally reversible, generally effects the exchange of extracellular Na+ for intracellular H+. It is currently believed that the Na+-H+ antiporter is regulated by a pH- or hydrogen ion-sensitive site on the cytoplasmic domain of the protein. This moiety is responsible for inactivating the transporter at a particular pH set point, thus keeping intracellular hydrogen ion concentration from falling to what would generally be a much lower equilibrium value (69,97). This pH-sensitive moiety also appears to be the site of phosphorylation resulting from the effects of hormones and growth factors (92), which may thereby change its set point and alter pHi. The gene encoding for the Na+-H+ antiporter has been cloned and the primary structure of the human antiporter protein recently determined (93). It appears to consist of 894 amino acids with multiple alternating hydrophobic and hydrophilic regions, which probably


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course back and forth from the cytoplasmic to the surface domains of the plasma membrane. Although the deduced amino acid sequence of the antiporter does not share significant homology with others previously described, its transmembrane configuration resembles that of the Cl--HCO; exchanger and other transport proteins, suggesting shared functional properties or, possibly, evolution from common precursors. The Na+-H+ antiporter is inhibited reversibly by the diuretic drug amiloride, which competes with sodium at the external face of the transporter (5,61). High concentrations of the drug (1 mM) are generally required to inhibit the antiporter in the presence of physiological sodium concentration, whereas sodium entry via epithelial sodium channels is generally blocked by concentrations that are several orders of magnitude lower. The recent synthesi s of amiloride analogues relatively specific for either sodium channels (benzamil) or the antiporter (dimethyl .amiloride) have th erefore greatly facilitated the study of sodium transport by these two mechanisms. Other inhibitors of the Na+-H+ antiporter include the alkaloids harmaline and quinidine, N-ethoxycarbonyl-Zethoxy-1,Zdihydroquinoline (EEDQ), and diethylpyrocarbonate. In addition, inhibitors of the vacuolar H+ATPase (see below), N-ethylameimide (NEM) and N,N’-dicyclohexylcarbodiimide (DCCD), may also inhibit the Na+-H+ antiporter (97). In general, all of these 6.6

0 Na+

No+

REVIEW

agents are either less specific than amiloride (and its analogues), irreversibly inhibit the antiporter, or both. Evidence for the Na+-H+ antiporter in epithelial cells and information concerning its kinetic properties can be derived from several different lines of experimentation. First, pHi can be measured as described above. Generally, cells or membrane vesicles are acidified, and recovery of pHi (or lack thereof) under different experimental conditions is monitored. Realkalinization mediated by the Na+-H+ antiporter is dependent on external Na+, is inhibited by amiloride, and is not affected by nor effects changes in transmembrane potential (i.e., is electroneutral). In addition, the antiporter is regulated by pHi such that it is inactivated at some physiological pHi (generally 7.0 under baseline conditions). Na+-H’ antiport was described in freshly isolated alveolar type II pneumocytes (79) utilizing pHi measurements made with the fluorescent dye BCECF. These findings were confirmed shortly thereafter in both isolated type II cells and alveolar pneumocytes in primary culture with the use of similar techniques (91). In the former study, freshly prepared pneumocytes were acidloaded by exposure to the H+-K+ ionophore nigericin in the presence of low [K+1,. Acidification results from exchange of intracellular K+ for extracellular H+, and can be maintained by washing the cells with bovine serum albumin to scavenge the ionophore. When cells No+ + VAL

Nat

+ AMIL

6.5

6.4

LA-

6.3 ’

6.4 hit i al pHi

1 min

>

L

7.2

Fig. 1. Evidence for Na+-H+ antiport activity in type II alveolar epithelial cells and dependence of Na’-H’ antiport on intracellular pH (pHi). A: freshly isolated alveolar type II cells were loaded with the pH-sensitive fluorescent dye 2’,7’-bis(2-carboxyethyl)-5(6)-carboxy-fluorescein (BCECF), acidified by incubation in nigericin in low [K’] medium, and resuspended in Na’- and HCO;-free buffer at pH 7.4. Cell suspension (10 ~1) was added to a cuvette that contained (pH 7.4) 140 mM trimethylammonium chloride (0 Na+) or 140 mM NaCl (Na’) or 140 mM NaCl and 2 pg/ml valinomycin (Na’ + VAL) or 140 mM NaCl and lOA M amiloride (Na’ + AMIL). These curves show no recovery of pHi in absence of Na+, maximal recovery in presence of Na+, no effect of valinomycin, and inhibition by amiloride. This electroneutral, Na+-dependent, amiloride-inhibitable recovery from intracellular acidification is most consistent with Na’-H’ antiport. B: BCECF-loaded cells were washed and suspended in medium composed of Na+-free medium containing 130 mM KCl, titrated to pH 6.4, 6.6, 6.8, 7.0, or 7.2 with 1.0 N HCl. To set pHi, nigericin (0.5 pg/ml) was added to suspension buffer and incubation proceeded for 5 min. Ionophore was subsequently extracted with bovine serum albumin. Cell suspension (10 pl), treated in this manner, was added to cuvette holding a solution with 140 mM NaCl and 1.0 mM KCl, titrated to pH 7.4 with 6.1 mM HEPES and 2.0 mM Tris base. These data indicate that Na’H’ antiport is inactive in alveolar pneumocytes at pHi > 7 (79).


INVITED

were acidified in sodium-free buffer, rapid cytoplasmic alkalinization occurred when they were exposed to sodium (Fig. 1). This response was inhibited >90% by 100 PM amiloride. The K+ ionophore valinomycin, expected to change the membrane potential of the cell, had no effect, indicating the electroneutrality of the system (Fig. 1). Li+ partially supported the realkalinization, whereas other monovalent cations (K+, Rb+, and Cs’) did not. Kinetic analysis (Hill equation analysis) for Na+ at the external binding site were consistent with a 1:l stoichiometry of Na+ and H+ for the antiporter. Taken together, these properties are all consistent with the presence of Na+-H+ antiport in alveolar pneumocytes. Another result of these studies was the determination of the dependence of proton flux on pHi. Antiport activity was detected at pHi values of 56.8, with no activity observed at pHi 7.0-7.2 (Fig. 1). This characteristic property of the antiporter implies that it may not be operative at baseline pHi (>7.0) and suggests that by modulation of its set point (e.g., by growth factors), it may contribute to regulation of pHi when alveolar pneumocytes are in a stimulated state. A second experimental technique for discerning the presence and characteristics of the Na+-H+ antiporter in epithelia is to measure fluxes of 22Na+ and other radionuclides. In a preliminary study, pHi was concurrently monitored using fluorescence (BCECF) and distribution of a weak acid ( [14C]DMO) in freshly isolated cells and short-term cultures of adult rabbit type II cells (37). The authors found a Na+-dependent alkalinization in the presence of the surfactant secretagogues terbutaline (a ,@-adrenergicagonist) and 12-0-tetradecanoylphorbol13acetate (TPA) (a phorbol ester). In parallel studies, measurement of 22Na+influx under similar conditions of stimulation showed an immediate increase in the uptake of Na+ after addition of either agent. Calculation of net influx of Na’ and efflux of H+ resulted in a stoichiometry of 0.881, most consistent with electroneutral Na+-I--I+ antiport. These findings led the authors to conclude that Na+-H+ antiport played a role in the transduction of secretory signals in the type II cell. More recently, a group of investigators have described sodium-proton exchange across apical membrane vesicles prepared from fetal sheep alveolar type II cells in studies utilizing 22Na+ flux measurements (19, 98). In these studies, evidence for apical Na’-H’ antiport was threefold. First, 22Na+ uptake into vesicles was augmented by an outwardly facing hydrogen ion gradient (PH. = 5.5 vs. pHoUt = 7.5) compared with control in the absence of such a gradient. Second, uptake in the presence of a proton gradient was inhibited by 1100 PM amiloride. Finally, Na+ uptake demonstrated a saturable component showing kinetics similar to other epithelial Na+-H+ antiporters. Taken together, these data are consistent with the presence of Na+-H+ antiport on the apical surface of fetal alveolar type II cells. Na’-H+ antiport has generally been described on the apical surface of epithelia, although Na+-H+ antiporters with different pharmacological characteristics have been described as coexisting on both apical and basolateral aspects (49) [or on the basolateral surface only (50)] of epithelial cells. They are probably of major importance

REVIEW

L5

for fetal lung biology and regulation of alveolar fluid pH (see below). Most recently, the regulation of the Na+-H+ antiporter was investigated in rat renal cortex using a [“P]DNA probe to antiporter mRNA (62). In this study, the authors found increased Na+-H’ antiporter mRNA in the tissue after 5 days of metabolic acidosis but no change after the same period of respiratory acidosis. These results suggest that the enhanced proximal tubule Na+-H+ antiporter activity and bicarbonate reabsorption observed in respiratory acidosis involve mechanisms other than increased Na+-H+ antiporter gene expression. This study indicates the utility of DNA probes for further study of the regulation of Na+-H+ antiport (and other pH;-regulating ion transport mechanisms) in epithelial cells at the molecular level. H+-ATPases. Several types of H+-ATPases have been described in epithelial cells. All of these bicarbonateindependent ion transport mechanisms directly utilize cellular energy by hydrolyzing ATP to effect extrusion of hydrogen ion. These pumps are either electrogenic and require interaction with a parallel ion conductance (e.g., K+ channel) to maintain electroneutrality, or involve the countertransport of another cation (e.g., the gastric H+, K+-ATPase). Three classes of H+-ATPases are generally described. The most structurally simple, consisting of only one or two subunits, are the El-E2 ATPases. This designation refers to their catalytic cycle, which includes an acyl phosphate intermediate, and is inhibited by vanadate (V04) acting as a substitute for phosphate. This group of ATPases includes the Na+,K+-ATPase, gastric H+, K+-ATPase, and Ca2+-ATPases. Inhibitors of these pumps include ouabain (Na+,K+-ATPase only) and omeprazole, a potent inhibitor of the gastric H+,K+-ATPase now in clinical use as an inhibitor of gastric acid secretion (71) A-second group of proton pumps, the FIFo variety, are more structurally complex, and are localized primarily in mitochondria, chloroplasts, and bacterial membranes. This group of ATPases normally operates in a reverse mode to synthesize ATP. Their designation refers to their structure, which consists of a hydrolytic/synthetic component (F,) and a hydrophobic transmembranous proton channel (Fo) sensitive to the inhibitor oligomycin (104). These pumps are sensitive to a large number of inhibitors, excluding vanadate and the water-soluble sulfhydryl reactive agent NEM. A third group of H+-ATPases found in epithelia have been designated vacuolar or vATPases. So designated because they are responsible for acidification of a variety of intracellular compartments, including endosomes and lysosomes (38), they share some structural properties and possible biochemical ancestry with the FIFo proton pumps. Vacuolar-type H+-ATPases have been described in the plasma membrane of both proximal and distal renal epithelia, where they contribute to urinary acidification. They have also been described in osteoclasts, where formation of an acidic extracellular compartment by these cells facilitates bone resorption (6). Although most frequently described in the context of transepithelial acid secretion, vacuolar H+-ATPases may also con-


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tribute to pHi regulation (51, 105). The vacuolar H+ATPases are insensitive to vanadate and oligomycin but are inhibited by the alkylating agent 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl) and by NEM and DCCD in micromolar concentrations. As is the case for Na+-H+ antiport, H+-ATPases must respond to a variety of intra .- and extracel lular influences. For example, renal tubular H+ secretion has been shown to respond to alterations in acid-base status, to hormones, and to potassium depletion (43). Several mechanisms whereby H+-ATPase activity is regulated are known. In the distal nephron, factors that increase the lumen negative potential in the collecting duct enhance proton secretion. Decreases in luminal pH decrease H+ secretion in a fashion likened to the slowin .g of an automobile going up a steep hill (43). Cells respond to changes in pHi and PCO~ by insertion of proton pumps into the cell membrane by Ca2+ -dependent exocytotic fusion (4, 20, 111). H+-ATPase expression in proliferating vs. quiescent epithelia is also regulated by some, as yet unknown, cellular regulatory mechanism. For example, nondividing cultured inner medullary collecting duct (IMCD) epithelia in the center of growing nests of cells express proton pumps, whereas dividing cells at the periphery of such nests express only Na+-H+ antiport activity (15). We have obtained evidence for active H+ secretion by rat alveolar epithelial cells grown in primary culture for 3-4 days in studies with the fluorescent probe BCECF (66) (Fig. 2). In these studies, pHi of alveolar epithelial cells was acutely lowered by NH3 prepulse in HCOT-free N- 2 - hydroxyethylpiperazine - N’ - 2 - ethanesulfonic acid

1

.-

Control

1 .o

0.8

._

0.6

Sk a 0.4

0.2

0.0 6.0

Time (min)

Fig. 2. Effects of amiloride and N-ethylmaleimide (NEM) on recovery from acute intracellular acidification in alveolar epithelial cells. Alveolar epithelial cells grown in primary culture were acidified by NH3 prepulse in HCOT-free buffer, and recovery of pHi monitored thereafter using the fluorescent probe BCECF. Recovery phases from typical experiments (66) are illustrated and recovery expressed as ApHi from the nadir vs. time (min). Under control conditions, cells were acidified in Na+-containing buffer without inhibitors present. pHi fell to a nadir of 6.42 and recovered with initial rate (dpHi/dt) of 0.22 min. In presence of amiloride (Amil), pHi fell to a nadir value of 6.50 and recovered with dpHi/dt of 0.09 min. In presence of NEM, pHi fell to 6.33 and recovered with dpHi/dt of 0.13 min. In presence of both amiloride and NEM, pHi fell to a nadir value of 6.32 and essentially did not recover. These data, showing partial inhibition of recovery by either amiloride or NEM and complete inhibition by both together, indicate the presence of 2 mechanisms of pHi recovery in alveolar epithelial cells, Na’-H’ antiport and H+-ATPase.

REVIEW

(HEPES)-buffered medium. To this end, cells were transiently exposed to 20 mM NH&l, resulting in intracellular alkalinization from influx of NH3 into the cell. Subsequent intracellular acidification occurs upon removal of extracellular NH&l due to efflux of NH3 out of the cell, with retention of impermeant NH: (9, 13). pHi recovery occurred in part by a mechanism that was Na’ independent and amiloride insensitive. This mechanism could be inhibited by depleting intracellular ATP (by exposure of the cells to KCN in the absence of glucose), implying the existence of an ATP-dependent process. It was also inhibited by NEM and DCCD, but not by vanadate or removal of extracellular K+, and could be inhibited in stepwise fashion by lowering pH,. These findings are most consistent with the presence of a vacuolar-type H+-ATPase in the plasma membrane of these alveolar epithelial cells. Its absence in freshly isolated cells (79) suggests that its expression may be anchorage dependent (see below). Evidence for the presence of a vacuolar H+-ATPase in lamellar bodies of alveolar type II cells has previously been described. Isolated lung lamellar bodies were found to maintain an acidic internal pH of 6.1 by measuring the distribution of the permeable basic amine [14C]methylamine across their limiting membranes (24). The pH gradient across the lamellar body membrane decreased when external pH, was decreased or when ATP was omitted and was inhibited by NEM and DCCD. These findings are consistent with active hydrogen ion secretion into lamellar bodies by a proton pump, most likely of the vacuolar type. In a later preliminary study, these investigators found that a subfraction of rat lung lamellar bodies contained a ouabain-insensitive ATPase activity inhibited in a dose-dependent fashion by NEM and DCCD (23). We have speculated that this may be the same mechanism as the plasma membrane proton pump, inserted into the membrane by fusion of lamellar body membranes during, for example, exocytosis of surfactant. Another group has published preliminary evidence for a K+,H+-ATPase in type II pneumocytes. These investigators found a potassium-dependent ATPase activity in apical membrane vesicles from guinea pig alveolar pneumocytes (14). Still others (17) have found preliminary evidence for ATP-dependent Na+-H+ antiport in alveolar pneumocytes, rather than active H+ secretion per se. Although it is difficult to completely reconcile these discrepancies at the present time, it is possible that species differences or experimental variation (the latter due in part to differences in the state of activation of the cells, as was the case for IMCD cells cited above) could account for some of them. Cl--HCO~ exchange. The Cl--HCO: exchanger (also referred to as the Na+-independent Cl--HCO, exchanger), like other HCOT-dependent transport mechanisms, has been shown to be involved in pHi, volume and osmolarity regulation, transepithelial acid-base transport, and the transport of CO2 between the peripheral tissues and the lungs (10, 39, 68). As is the case for the Na+-H+ antiporter, the Cl--HCO, exchanger is driven by the gradients for Cl- and HCO; across the cell mem-


INVITED

brane. The exchanger normally mediates the one-for-one exchange of extracellular Cl for intracellular HCO;, an inward gradient for the former driving extrusion of the latter from the cell. The epithelial Cl--HCO; exchanger is structurally and functionally similar to the erythrocyte anion exchanger, designated the band 3 protein because of its position in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Na+-independent Cl--HCO: exchanger is inhibited by the stilbene derivatives 4,4’-diisothiocyanostilbene-2,2’-disulfonate (DIDS) and 4-acetamido-4’isothiocyano-stilbene-2,2’-disulfonate (SITS). Other HCOT-dependent ion transporters, including the Na+dependent Cl--HCO; exchanger (also referred to as a Na/HCO&l/H exchanger), which has not been described in alveolar pneumocytes, share this sensitivity to DIDS and SITS. They differ in that the Na+-dependent exchanger has an absolute requirement for extracellular sodium, which drives bicarbonate into the cell in exchange for chloride. Na+-dependent Cl--HCO: exchange is therefore primarily a mechanism for intracellular alkalinization, whereas Na+-independent Cl--HCO; exchange primarily effects excretion of base equivalents from the cell. Inasmuch as disposal of accumulated intracellular acid appears to be the primary imperative of pHi regulation, it may not be immediately apparent why a base-exporting Cl--HCOg exchanger should be important for epithelial cell homeostasis. In fact, Cl--HCO; exchange operating in this mode has been shown to modulate pHi (llO), acting in concert with other pHiregulating ion transporters. Similarly, for example, many epithelia employ a Cl--HCO: exchanger in parallel with a Na+-H+ antiporter to effect salt (and water) transport without net transport of acid or base (32). Although the cellular mechanisms regulating Cl--HCO; exchange are not as well known as those regulating Na+-H+ antiport, the exchanger’s activity is known to be modulated by pHi, hormones, and growth factors (42, 94). Cl--HCOy exchange was identified in rat alveolar epithelial cell monolayers grown in primary culture using fluorescence measurements of pHi made with BCECF (Fig. 3) (80). Under steady-state conditions in the presence of 25 mM 95% HCOg-5% Con at pH, 7.4, baseline pHi was unaffected by the removal of extracellular sodium. In contrast, a DIDS-inhibitable rise in pHi was observed in Cl--free buffer of otherwise identical composition, suggesting that Cl--HCO: exchange contributes to pHi regulation under baseline conditions. In other experiments, the cells comprising the monolayer were acutely alkalinized by transient exposure to HCO:-C02. To this end, the cells are briefly exposed to medium buffered by 25 mM 95% HCOg-5% COZ, which results in acidification due to entry of freely permeant COa into the cells. When this medium is replaced by one of identical composition buffered by HEPES, a rapid rise in pHi ensues due to efflux of C02, but not impermeant HCO;, from the cell. Recovery from this alkaline load required extracellular Cl- (Michaelis constant = 11 mM) and was inhibited by 60% in experiments performed in the presence of DIDS. Recovery from an alkaline load was not affected by the presence or absence of extracellular Na+.

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7.8 i-$l-c

7.0

7.2 I

?.21-

1 min

Fig. 3. Evidence for Cl--HCO, exchange in alveolar epithelial cells: recovery of pHi from an alkaline load in presence of various anions. Monolayers of alveolar epithelial cells grown in primary culture were loaded with BCECF and incubated in HCOT-containing medium equilibrated with 5% CO, to pH 7.4. Cells comprising the monolayer were alkalinized by replacement of the buffer with one of similar composition without HCOJCO,. A.- bicarbonate buffer was evacuated and replaced by HCO;-free buffer containing Cl-. B-D: Cl- in A was replaced isosomotically with gluconate (B), sulfate (C), or isethionate (D). These data showing reacidification (recovery) in presence, but not absence, of external chloride in presence of HCO: are consistent with presence of Cl-- HCO; exchange pathway in alveolar pneumocytes (80).

Taken together, these findings demonstrate that Na+independent Cl--HCO: exchange modulates pHi and enables the cell to recover from an alkaline load in these cultured alveolar pneumocytes. We have also recently obtained additional preliminary evidence for Cl--HCO: exchange in alveolar epithelial cells by immunocytochemical localization using polyclonal antiserum to murine band 3 protein (courtesy of S. Alper) (unpublished observations). Alveolar epithelial cells at day 3 in primary culture were incubated with polyimmune serum or normal rabbit serum (control) and then incubated with FITC-goat anti-rabbit antibody and viewed by phase and epifluorescence optics. All cells were diffusely reactive with this serum, and controls were uniformly negative. These findings further support the presence of Cl--HCO; exchange in alveolar pneumocytes and suggest the utility of this approach for identifying other ion transporters and defining their polarized distribution in these cells. Na’-HCO, cotransport (symport). The electrogenic Na+-HCO, cotransporter (symporter) is also a HCOTdependent ion transport mechanism that contributes to the regulation of pHi in both epithelial and nonepithelial cells. This transporter effects the coupled transport of Na+ and HCO; (and/or other related ionic species, e.g., CO:-) either into or out of the cell, thus operating in either an acid loading or acid extrusion mode. First described in the renal proximal tubule of the tiger salamander Ambystoma tigrinum in 1983 (ll), it was shown to be an electrogenic process, implying a HCO$Na+ stoichiometry different from 1:l. Subsequent studies in mammalian proximal tubule preparations have established a 3:1 stoichiometry for these cells in which Na+HCO: cotransport operates in the acid-loading mode (12,


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86), whereas this ratio appears to be G!:l in some other cell types in which it effects Na+ and HCO; influx (i.e., net acid extrusion) (57). It is therefore apparent that the magnitude and directionality of Na’-HCO: cotransport can be affected by the membrane potential and transmembrane gradients for both Na+ and HCO;, as demonstrated directly (101) in basolateral membrane vesicles from rabbit renal cortex. Na+-HCO; cotransport has been identified in many different epithelial tissues but has been studied most extensively in the renal proximal tubul .e. At this site, a basolateral cotransporter contributes to the reabsorption of filtered bicarbonate (12, 86). As is the case for the other pH;-regulating ion transporters previously described, Na’-HCOT cotransport contributes to regulation of pHi and electrolyte composition and/or transepithelial transport in different types of cells. Its activity is modulated by soluble factors (e.g. hormones), and basolateral Na+-HCO; cotransport in the kidney has also been shown to be increased (or decreased) in parallel with apical Na+-H+ antiport in the presence of chronic respiratory acidosis (or alkalosis) (90). No unique inhibitor of the Na+-HCO; cotransporter is known (12). It is inhibited by DIDS and SITS in epithelia, although some nonepithelial cotransporters are insensitive to these agents (59). Criteria for identifying an electrogenic Na’-HCO; cotransporter based on isolated measurements of pHi intracellular Na activity ( aNa), or Vm have recently been reviewed (12). To this end, several methods for demonstrating electrogenic cotransport of both Na+ and HCO; exchange, independent of Cl- and other ions and inhibitable by DIDS or SITS, are described. The authors caution that, particularly insofar as inhibitors are of limited utility in identifying the cotransporter, particular care must be exercised in distinguishing it from similar ion transport mechanisms (e.g., Na+-dependent Cl--HCO; exchange). We have recently shown evidence for Na+-HCO; cotransport in alveolar epithelial cells grown in primary culture using pHi measurements with the fluorescent dye BCECF (67) (Fig. 4). In these studies, alveolar epithelial cell monolayers were acidified by incubation in HCOyfree medium followed by replacement with a HCOy-COabuffered medium of otherwise similar composition (CO2 pulse). Rapid intracellular acidification ensues due to influx of COz while the relatively impermeant HCO; is excluded from the cells. The rate of recovery from intracellular acidification was reduced by removal of extracellular Na+ but not extracellular Cl-. Recovery was also inhibited by DIDS (500 ,uM) but not by amiloride (1 mM). In parallel experiments, pHi was acutely lowered in the presence of HCO, by NH3 prepulse (see H+ATPases). Again, the rate of recovery was reduced in the absence of extracellular Na+. These data indicate the presence of a Na+-dependent, Cl--independent, DIDSsensitive, and amiloride-insensitive mechanism of recovery from acute intracellular acidification in alveolar pneumocytes, most consistent with Na+-HCO; cotransport (symport) effecting acid extrusion under these experimental conditions. Similar to the other ion transport mechanisms described above, this symport mechanism may contribute to regulation of pHi in alveolar pneu-

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X

d”

0.2

0.1

6.0

Time (min)

Fig. 4. Effects of amiloride, 4,4’-diisothiocyanostilbene-2,2’-disulfonate (DIDS), and removal of extracellular Na’ on recovery from intracellular acidification in alveolar epithelial cells. Alveolar epithelial cells grown in primary culture were incubated in HCO;-free medium and subjected to intracellular acidification by exposure to COJHCO; (CO2 pulse). pHi during recovery (i.e., realkalinization) was monitored using BCECF. Recovery phases from typical experiments (67) are illustrated, and recovery is expressed as ApHi from the nadir vs. time (min). Control data are from experiments performed in Na’ buffer without inhibitors present. Under these conditions, pHi fell to a nadir of 7.02 and recovered with an initial rate (dpH/dt) of 0.16 min? In presence of amiloride, pHi fell to a nadir of 7.01 and recovered with dpHi/dt of 0.16 min. In presence of DIDS, pHi fell to 7.04 and recovered with dpHi/dt of 0.07 min. In absence of extracellular Na+, pHi fell to a nadir of 7.00 and recovered with dpHi/dt of 0.07 min. These results demonstrate a Na+-dependent, amiloride-insensitive, DIDS-inhibitable pathway for net acid extrusion in HCOF-medium consistent with Na’HCO; symport (cotransport) in alveolar epithelial cells.

mocytes, transepithelial transport of acid/base equivalents across the alveolar epithelium, and modulation of pH of alveolar fluid in adult mammalian lungs. Baseline pHi in alveolar pneumocytes. It should be noted that several different values for baseline steadystate pHi have been published for alveolar pneumocytes. The baseline pHi of isolated alveolar type II cells has been reported to be 7.07 (79) and 7.36 (91) from rat and 7.22 (37) from rabbit (pH,, 7.4). Baseline pHi in cultured rat alveolar epithelial cells has been reported to be 7.14 (17), 7.19 (80), 7.48 (91), and 7.50 (66) in the absence of HCO; and 7.32 (80) or 7.4 (67) in the presence of HCOg-C02. Differences in experimental conditions aside, these apparent discrepancies may possibly be accounted for in several ways. First, elevations in baseline pHi associated with cell attachment and spreading have been described in a variety of cell types (72,96). Second, cells may exhibit higher, lower, or unchanged baseline pHi in the presence or absence of HCO: (80), reflecting variability in the presence and cellular distribution of HCOY-dependent and HCOT-independent pHi-regulating ion transport mechanisms among different cells. Finally, the observed difference in pHi may reflect differences in the degree to which the alveolar epithelial cells in the different studies have been stimulated by (as yet) unknown factor or factors. Although baseline pHi values are generally reported to be 7.0-7.4 in quiescent cells (88), changes in pHi mediated by alterations in the set point of the Na+-H+ antiporter or changes in the expression or distribution of one or more ion transporters may accompany metabolic or development changes (47).


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Taken in this context, it is understandable why these different values for baseline steady-state pHi in alveolar epithelial cells may have been observed. SIGNIFICANCE

OF

INTRACELLULAR

PH

As noted in our introductory remarks, pHi and its regulation and effects have many implications for eucaryotic cell function and biology. For example, pHi modulates the activity of intracellular enzymes, contractile elements and cytoskeletal proteins, and intracellular ion activities and ion conductances. Furthermore, changes in pHi appear to exert effects on control of the cell cycle, a concept supported by the documented stimulatory effects of growth factors on Na+-H+ antiport and the other pH;-regulation ion transporters (47). These effects have been summarized in several recent reviews (18, 75). The effects of pHi on surfactant secretion by alveolar type II cells have been studied by several investigators. As noted above, pHi regulation was investigated in freshly isolated rabbit alveolar type II cells using both the fluorescent dye BCECF and the weak acid DMO (37). Addition of surfactant secretagogues [terbutaline (10 PM) or TPA (100 nM)] resulted in an intracellular alkalinization from pHi 7.22 to 7.33 in the presence of Na+, whereas no change in pHi was observed in the absence of Na+. Treatment of cells with inactive analogues of TPA that do not induce surfactant secretion did not alter pHi. The ,&agonist inhibitor propranolol diminished terbutaline-induced alkalinization, and amiloride (200 PM) inhibited both the terbutaline- and TPAinduced elevations of pHi. Measurements of 22Na+ influx under similar conditions of stimulation demonstrated an increase in Na+ uptake in the presence of terbutaline or TPA. These findings led the authors to conclude that Na+-H+ antiport has a role in the transduction of secretory signals in type II pneumocytes. Conversely, others found no effect of terbutaline or TPA on pHi in rat alveolar pneumocytes at 22 h in primary culture (91). Inhibition of Na+-H+ antiport by the addition of amiloride or removal of extracellular Na+ did not reduce stimulated phosphatidylcholine secretion. These findings led the authors to conclude that pHi regulation in rat alveolar type II cells is mediated in part by an amiloride-sensitive Na+-H+ antiporter but that this system was not involved in terbutaline- or TPAinduced pulmonary surfactant secretion in primary culture. Although other factors may account for these negative findings, it is possible that the higher baseline pHi observed in this study (7.48) vs. that observed in the other (7.22) (37) may have precluded the secretagogueinduced alkalinization found in the latter study. More recently, secretion of [methyl-3H]choline-labeled lung surfactant phosphatidylcholine (PC) was investigated using an isolated rat lung preparation (21). In these studies, surfactant secretion was measured as total lung lipid radioactivity (>95% PC) recovered in cell-free lavage fluid. The author observed increased PC secretion when terbutaline (50 PM), 8-bromoadenosine 3’,5’-cyclic monophosphate (&BrcAMP, 100 PM), phorbol 12-myristate 13-acetate (30 ng/mg), or ATP (1 mM) were added to the perfusate. Secretion of PC was also increased in a

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time- and CO2 concentration-dependent fashion if the lungs were ventilated by 0% CO2 rather than 5% or 10% C02. This increase was reversed with addition of diffusible weak acids (acetate or butyrate, 25 mM) to the perfusate. As noted in this study (21), the relative increase in surfactant secretion observed during low CO2 ventilation was not found to be reversed with 0.1 N HCl by others in dog lungs (99). PC secretion was also increased when the lungs were perfused with NH&l (10 mM), suggesting that intracellular alkalosis, as might occur during hyperventilation, stimulates lung surfactant secretion. Although the results were similar to those of the first study cited above (37) in many respects, Chander (21) found that alkalosis-stimulated secretion of PC was additive to that with terbutaline or &BrcAMP, implying that the effects of alkalosis on surfactant secretion were not mediated by the ,&adrenergic pathway. Chander also found that the effects of alkalosis on surfactant secretion could be blocked by inhibitors of protein phosphorylation, chlorpromazine (1 mM), or 1,5- (isoquinolinylsulfonyl)-2-methyl-piperazine (15 PM). Taken together, these results led to the conclusion that alkalosis-induced surfactant secretion in alveolar type II cells could be mediated via activation of protein kinase C or some other mechanism different from that responsible for ,&agonistor hyperinflation/stretch-induced surfactant secretion. Evidence has been presented that terbutaline-induced secretion may occur due to activation of an adenosine 3’,5’-cyclic monophosphate (CAMP)-dependent protein kinase (22), while the stretch-induced secretion is related to calcium mobilization, which occurs after a single mechanical stretch of type II cells (117). In a more recent preliminary study, this line of investigation was continued by studying the effects of alkalosis on surfactant secretion in cultured alveolar pneumocytes (25). The authors measured the secretion of [3H]PC by alveolar epithelial cells in KRB (i.e., HCO; containing) buffer containing 25 mM HEPES at pH, 7.4 in 5% CO2 in air after overnight culture. PC secretion was stimulated by ATP (1 mM) and PMA (50 rig/ml). Intracellular alkalosis induced by NH&l (10 mM) or incubation in 0% CO2 stimulated secretion, whereas raising pH, from 7.4 to 7.8 did not. As observed in the intact lung study, inhibitors of protein phosphorylation dibucaine (100 PM), trifluperazine (10 PM), or H-7 (10 PM) blocked alkalosis-stimulated secretion. When phosphorylation of cellular proteins was determined using [T-~~P]ATP labeling, increased phosphorylation was apparent in proteins migrating at 45, 36, and 25 kDa after preincubation of cells for 2 h in 0% vs. 5% C02. These preliminary observations provide additional evidence that alkalosisstimulated secretion of surfactant is associated with activation of protein kinases to increase protein phosphorylation. Evidence exists for other roles for pHi and the ion transport mechanisms by which it is regulated in lung cells. Changes in pHi have been noted to affect conductantes for K+, Na+, Cl-, and other ions in a wide variety of epithelia (64, 82) and excitable cells (40). Although experimental data are lacking thus far for the alveolar epithelium, preliminary data support the hypothesis that


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pHi modulates transmembrane ion conductances in airway epithelia (2, 45). Furthermore, pHiregulating ion transporters may contribute to transepithelial transport in these cells (lo@, as they do in other epithelial cells (32) The ion transport mechanisms involved in the regulation of pHi in alveolar (and airway) epithelial cells may similarly modulate the extracellular milieu. Adaptations of microelectrode techniques were used to measure pH of the aqueous subphase of the alveolar lining fluid in anesthetized adult rabbit lungs (78). To measure pH, a H+-sensitive glass electrode with a 5-7 pm tip was constructed and used to impale subpleural alveoli from the serosal (pleural) side. Alveolar subphase fluid was found to be acidic, as previously noted in fetal lambs (l), with a pH of 6.92 when the extrapleural surface buffer and arterial pH were 7.4. Extracellular alveolar fluid pH remained constant as the surface buffer pH was varied between 6.5 and 7.5. These results led to the conclusion that alveolar subphase fluid pH was regulated, possibly by active transport of acid-base equivalents. Although their result (pH 6.92) was significantly different from that of plasma and that measured in the alveolar fluid of the fetal lamb (pH 6.27) (l), it agreed closely with the pH of airway surface liquid (ASL) secreted by ferret trachea in vitro (6.85) (63). As was also the case for alveolar subphase fluid pH measurements, varying external organ bath pH resulted in proportionally smaller changes in airway surface liquid pH. These results suggest that there may be active control of both ASL and ALF pH, presumably based on active secretion of H+/ HCO;. The importance of the formation and retention of liquid within the lumen of the fetal lung for lung growth and development in utero is well established (7). The significance of fetal lung fluid composition (including pH), however, is unknown. It has been suggested that low ALF pH could affect, for example, the activation and inhibition of enzymes, the recycling of surfactant, and the function of alveolar macrophages (78). Others have speculated that low ASL pH could operate to limit bacterial adhesion and growth and might affect ciliary function in the airways (63). These and other potential roles for low surface pH require further exploration. The regulation and effects of pHi may ultimately have other important implications for the function and biology of alveolar pneumocytes. Changes in pHi may affect the barrier properties of the alveolar epithelium, critical for the maintenance of normal alveolar fluid balance and prevention of pulmonary edema. Changes in pHi have been implicated in modifications in cytoskeletal proteins and cell junctions (56, 58, 114), both important for the formation of a functional epithelial barrier. pHi may also affect macromolecule traffic within and across cells (30). The effects of pHi on protein transport, necessary for the resolution of pulmonary edema resulting from increased epithelial permeability, may therefore also be important. pHi may affect cell survival from ischemic injury. Intracellular acidosis has been reported to protect against the onset of cell death after ATP depletion in cultured rat hepatocytes (46). The fall in pHi observed

REVIEW

in cultured alveolar pneumocytes after ATP depletion (66) may serve a similar purpose. In a whole organ study using NMR measurements in isolated blood-perfused pig lungs, a value for tissue pH of 7.47 under normoxic conditions was obtained (84). After 15 min of ischemia, ATP declined by >90% and pH fell to 6.37, recovering after IO min of reperfusion to 7.21. These findings are qualitatively similar to those described for anoxia in heart (116) and other organs. Similar measurements described in a preliminary report (53) show a much lower value for the pH of the degassed rat lung (7.0), with a smaller decrease (0.2 pH units) after 30 min without perfusion. Consideration of pHi and cell survival may prove to be important for organ preservation for lung transplantation. This relationship has already inspired preliminary work on novel cancer chemotherapy agents that inhibit pHi regulating mechanisms, thereby altering pHi (and neoplasic cell survival) in tumors (89). Ischemic injury has also been shown to induce loss of polarity in some epithelia (73), inevitably resulting in impaired transepithelial transport of acid/base equivalents, salt, and water. Cytotoxic edema occurring during reperfusion after ischemia or during the recovery from diabetic ketoacidosis has been attributed to stimulation of Na+-H+ antiport, resulting in rapid Na+ uptake and cell swelling (47). Similar processes could be operative in the formation of alveolar edema in the lung injured by ischemia-reperfusion or by other mechanisms. Finally, changes in pHi may signal changes in growth or differentiation in both epithelial and nonepithelial cells. Increased pHi has been correlated with augmented DNA synthesis, changes in enzyme activities, and other functional changes likely to result in cell proliferation and/or differentiation (55, 77, 81). A role in cellular proliferation has been found for the Na+-H+ antiporter and growth factor-induced cytosolic pH changes (47). For example, one group of investigators isolated mutant Na+-H’ antiporter-deficient fibroblasts and demonstrated their inability to initiate growth factor-induced cell division or DNA synthesis at neutral or acidic extracellular pH (85). These results imply that intracellular alkalinization is necessary, though possibly not sufficient, to induce proliferation in these cells. Similarly, mammalian cells transfected with a yeast H+-ATPase gene express this ion transporter, show increased pHi, and acquire tumorigenic properties, suggesting that this gene may behave as an oncogene in animal cells (83). Similar effects in alveolar epithelial cells would have important implications for type II cell to type I cell differentiation and for carcinogenesis in the lung. SUMMARY

AND

FUTURE

DIRECTIONS

Experimental evidence supports the concept that pHi in alveolar pneumocytes is regulated by the cell, effected by several different ion transport mechanisms present in the plasma membrane. Although this appears to be true for all epithelia, several aspects related to pHi regulation in the alveolar epithelium may ultimately prove to be distinctive. These are likely to include the polarized distribution of the ion transport mechanisms under baseline and stimulated conditions, the specific mechanisms


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by which these transporters are regulated, the contributions of these transporters to other cell functions (e.g., transepithelial transport, volume and osmolarity regulation), and the funcionality of pHi as a mechanism for signal transduction (e.g., surfactant secretion) and of the pH;-regulating ion transporters as modulators of the extracellular alveolar milieu. The question of the distribution of pHi-regulating ion transporters with respect to membrane polarity across the alveolar epithelium has only recently begun to be addressed. The cellular mechanisms of transepithelial acid-base transport across the alveolar epithelium cannot be understood, nor can a general scheme of pHi regulation in alveolar pneumocytes be deduced without this information. In experiments by Butcher et al. (19) and Shaw et al. (98) the presence of Na+-H+ antiport was demonstrated in apical vesicles prepared from alveolar pneumocytes from fetal lambs. As noted previously, this finding does not exclude the possibility of basolateral Na+-H+ antiport in fetal alveolar pneumocytes and cannot necessarily be applied to the adult alveolar epithelium. It is, however, an example of one approach that can be used successfully in alveolar epithelial cells for determining transport properties of apical vs. basolateral epithelial membranes (70). Several other experimental approaches may be helpful, particularly if applied to a recently developed model of alveolar epithelium in culture in which pneumocytes are grown in primary culture on tissue culture-treated permeable supports (28). This system is well suited for the study of alveolar epithelial polarity, since the cells form electrically tight monolayers (Z&O00 Q. cm2) after 2-3 days under these conditions. These monolayers can then be used to functionally separate apical and basolatera1 fluid compartments in vitro in a fashion analogous to an Ussing chamber. One approach to assessingpolarity could be to measure pHi with BCECF or other fluorescent pH-sensitive probes in these monolayers and to differentially manipulate separate apical and basolateral fluid compartments with respect to composition and transport inhibitors. A similar approach could be used to determine changes in intracellular ion activities (e.g., Na’, K+, and Cl-) utilizing newly developed fluorescent probes (26,44,52,112). This type of study has been used successfully in several types of renal epithelia to determine the polarity of ion transporters in these tissues (50) and has been adapted for pHi measurement in single cells with the use of microspectrofluorometry (74). Another way to assessepithelial polarity of ion transporters (and determine their stoichiometry) using tight epithelial monolayers is to measure radioisotope uptake from apical and basolateral compartments. As noted previously, this approach has been used in a similar system to demonstrate the presence of Na+-H+ antiporters having different pharmacologic characteristics (i.e., different sensitivity to amiloride) on the apical and basolateral aspects of LLC-PKIA monolayers (49). A different approach, which can be used in cultured epithelia as well as in situ, is immunocytolocalization with either monoclonal antibodies or polyclonal antisera raised against the specific ion transport proteins or their subunits. Antibodies have been raised against many of

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the pH;-regulating ion transport mechanisms, including the Na+-H+ antiporter (93), Cl--HCO, exchanger (35, 54), vacuolar H+-ATPase (16), and H+,K+-ATPase (107), and the epithelial polarity of some of these transporters has been determined by immunofluorescence and/or immunoelectronmicroscopy using these mechanisms. For example, polyclonal antibodies raised against subunits of purified bovine kidney plasma membrane (vacuolar) H+ATPase were used in one study to localize a plasma membrane H+-ATPase in rat kidney intercalated cells in situ (16). The authors were able to label apical proton pumps using this antibody and were also able to show that adjacent cells displayed reversed polarity in some cases (i.e., basolateral pumps only). Because these techniques are adaptable for use in cultured epithelium as well as for in situ lung preparations, transporter polarity can be established for different cell types (i.e., type I vs. type II cell) and at different developmental stages (see below) or during recovery from injury. Determination of the polarity of pHi-regulating ion transport mechanisms in alveolar pneumocytes may provide the background by which several more complex issues can be investigated. One such issue is the expression of polarized membrane proteins by these cells at different metabolic states. For example, pH;-regulating ion transport mechanisms in renal cells have been shown to undergo changes in polarity during acidosis (95). Considering the lung’s greater importance as an acid-excreting organ, such changes would not be unexpected in the alveolar epithelium as well. Furthermore, these transport mechanisms undergo changes in expression at various developmental stages. For example, Na+-H+ antiport has been used as a marker of development and polarization during reorganization of LLC-PKIA cells into an epithelial membrane (113). Determination of the polarity of pH;-regulating ion transport mechanisms in alveolar pneumocytes may therefore also be useful toward pursuing the issue of cellular heterogeneity in the alveolar epithelium. In particular, these ion transport mechanisms may be different in type I and type II cells, and their presence and distribution may change in a characteristic fashion when type II cells differentiate into type I cells. It is presumed, although not yet proven, that the transport properties described for freshly isolated type II cells reflect type II cell properties in vivo. Cultured alveolar pneumocytes, on the other hand, may exhibit transport (and other) properties common to or intermediate between type I and type II cells, reflecting their relative degree of differentiation or dedifferentiation in vitro. Although type I cells comprise most of the alveolar epithelial surface, it has not yet been possible to purify and grow them in primary culture, making it difficult to study the transport (and other) properties of these cells directly. The cultured alveolar epithelial cells utilized in a new model of the alveolar epithelium described above, in which electrically tight cell monolayers are grown on permeable supports, appear to display a type I cell-like phenotypic morphology (27). Although we cannot yet identify these cells unequivocally as type I cells, recent experiments with type I cell monoclonal antibodies (33, 34) suggest that they may express, at least in part, type


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I cell phenotype. This model of alveolar epithelium may be useful to help determine type I cell transport properties and pHi regulation. Finally, the regulation and effects of pHi may ultimately have other important implications for the function and biology of alveolar pneumocytes. Changes in pHi may affect the barrier properties of the alveolar epithelium, critical for the maintenance of normal alveolar fluid balance and prevention of pulmonary edema (31). Changes in pHi have been implicated in modifications in cytoskeletal proteins and cell junctions (56, 58), both important for the formation of a functional epithelial barrier. pHi may also affect macromolecular traffic within and across cells (30). The effects of pHi on protein transport, necessary for the resolution of pulmonary edema states resulting from increased epithelial permeability, may therefore also be important. Finally, and perhaps most significantly, changes in pHi may result in changes in the growth, proliferation, differentiation, or neoplastic transformation of both epithelial and nonepithelial cells. It is therefore reasonable to consider that pHi could modulate processes such as type II cell differentiation in the alveolar epithelium. In summary, evidence has been presented in favor of the hypothesis that pHi in alveolar pneumocytes is regulated by several plasma membrane ion transport mechanisms capable of effecting acid/base entry or extrusion from the cells. It appears likely that pHi is regulated in these cells by multiple intra- and extracellular factors and that pHi, in turn, affects a wide spectrum of cellular processes (e.g., surfactant secretion). It is expected that future investigation will reveal further important effects of the regulation of pHi on the function and biology of the alveolar epithelium. This work was supported in part by the American Lung Association of California, the New York Lung Association, the Cystic Fibrosis Association of Greater New York, American Heart Association Grantin-Aid 89-749, and National Heart, Lung, and Blood Institute Research Grants HL-38578, HL-38621, and HL-38658. R. L. Lubman is a Parker B. Francis Fellow in Pulmonary Research. Address for reprint requests: R. L. Lubman, Div. of Pulmonary and Critical Care Medicine, University of Southern California GNH ll900, 1410 Biggy St., Los Angeles, CA 90033. REFERENCES 1. Adamson, T. M., R. D. H. Boyd, H. S. Platt, and L. B. Strang. Composition of alveolar liquid in the foetal lamb. J. Physiol. Lond. 204: 159-168, 1969. 2. Al-Bazzaz, F. J., and A. Koutsouris. Changes in intracellular acid-base milieu affect membrane transport in bovine trachea (Abstract). Am. Rev. Respir. Dis. 137: A220, 1988. 3. Aronson, P. Identifying secondary active solute transport in epithelia. Am. J. Physiol. 240 (Renal Fluid Electrolyte Physiol. 19): Fl-Fll, 1981. 4. Arruda, J. A. L., G. Dytko, and Z. Talor. Stimulation of H’ secretion by CO, in turtle bladder: role of intracellular pH, exocytosis, and calcium. Am. J. Physiol. 258 (Regulatory Integrative Comp. Physiol. 27): R222-R231, 1990. 5. Benos, D. J. Amiloride: a molecular probe of sodium transport in tissues and cells. Am. J. Physiol. 242 (Cell Physiol. 11): C131C145, 1982. 6. Blair, H. C., S. L. Teitelbaum, R. Ghiselli, and S. Gluck. Osteoclastic bone resorption by a polarized vacuolar proton pump. Science Wash. DC 245: 855-857,1989. 7. Bland, R. D. Lung epithelial ion transport and fluid movement

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Intracellular pH regulation in the embryonic chicken lens epithelium.

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SV40T-immortalized lung alveolar epithelial cells display post-transcriptional regulation of proliferation-related genes.