1Inologic Inc., Seattle, Washington; 2University of North Carolina, Chapel Hill, North Carolina; 3European Molecular Biology Laboratory, Heidelberg, Germany; 4University of California, Davis, California; and 5Children's Hospital, Seattle, Washington
Submitted 2 December 2004 ; accepted in final form 22 April 2005
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ABSTRACT |
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epithelial Na+ channels; fluid absorption
In CF airway epithelia, ENaC-mediated Na+ absorption is increased 100300% (2). Initially, this was attributed to a direct molecular interaction between CFTR and ENaC. However, no such association was found and is unlikely, because the relationship between ENaC activity and CFTR function is reversed in the sweat glands. Recent studies have demonstrated that electrical potential (34), intracellular Cl concentration (24), or electrical coupling between Cl and Na+ fluxes (16) could all mediate the inhibition. Therefore, the agents that modulate non-CFTR Cl channel activity, such as Ca2+-activated Cl channels in CF tissue, could secondarily regulate ENaC.
Inhibition of Na+ flux through ENaC is predicted to completely compensate for the CFTR defect in restoration of airway surface liquid (ASL) in a mathematical simulation of airway epithelial cells (36, 37). In support of this, amiloride, an extracellular blocker of ENaC, temporarily increased mucociliary clearance in clinical trials (48). Problematically, the duration of amiloride action in vivo is very brief (1530 min), thereby limiting its therapeutic efficacy. This transience is due in part to the diffusion and dilution of effective concentrations of extracellular amiloride. The relatively short half-life of its target, ENaC, in the plasma membrane (40) may also contribute to its brief duration of action.
Some of the >30 naturally occurring inositol polyphosphates (IPs) and phosphoinositides (18, 28, 32, 42) are ion channel regulators (5, 1215, 18, 19, 21, 35, 39, 5052). Whereas D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], a trigger for [Ca2+]i release, is the most widely recognized (17), inositol tetrakisphosphates also modulate ion channel activities, including Cl and K+ channels (12, 18, 21, 50). A particular inositol phosphate, Ins(3,4,5,6)P4, targets Cl channels in epithelia (5, 13, 14). IPs are highly charged, thereby making them unlikely to cross membranes. Thus membrane-permeant forms have been developed that enable intracellular delivery, which recapitulates the ion channel modulating activity of endogenous IPs (27, 41, 50).
In contrast to extracellularly acting agents directed against the extracellular domain of ion channel pores, membrane-permeant IP analogs modulate ion channel activities from inside the cell. Analogs of slowly metabolized IPs can have an extended duration of action. Therefore, they have the potential for prolonged activity in contrast to the more rapidly eliminated extracellularly active compounds. We previously observed that direct application of a membrane-permeant analog of Ins(3,4,5,6)P3, INO-4995 (Fig. 1), to human nasal airway epithelia reduced the amiloride-inhibitable basal short-circuit current (Isc ) (49). This effect was long lasting but reversible after 48 h. In the current study, we investigated the ionic basis for the inhibition of Isc and probed whether it corresponded to therapeutically meaningful changes in fluid flux as assessed from open-circuit fluxes of 22Na. Thus, in the absence of INO-4995, the mucosa-to-serosa flux of 22Na across CF airway epithelia was inhibited by amiloride, but this amiloride sensitivity was abolished by INO-4995. INO-4995 had no effects on Na+ fluxes across non-CF airway epithelia. These effects of INO-4995 corresponded to slowing of the fluid absorption rate that portend therapeutic efficacy in CF. In related studies in a mouse nasal potential difference (PD) model, INO-4995 lowered abnormal nasal PD in gut-corrected CF mice (8), further substantiating its therapeutic potential.
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METHODS |
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CF human nasal epithelial (CFHNE) and non-CF nasal epithelial (HNE) cell cultures were cultured and prepared for Ussing studies as described elsewhere (49).
Monolayer preparation for Ussing studies. Epithelial cells (passage 2 or 3) were prepared for Ussing chamber and fluid transport studies using Snapwell permeable supports (0.4 µm pore size; Corning Costar, Cambridge, MA) coated with 1 µg/cm2 Vitrogen. Cells were plated at 105 cells/cm2 in keratinocyte serum-free media. After 2 days, the media were changed to bronchial epithelial growth medium (BEGM), a 1:1 mixture of DMEM (MediaTech/Cellgro, Herndon, VA), and bronchial epithelial basal media (BEBM) (Clonetics/Biowhittaker, Walkersville, MD), with the following supplements: hydrocortisone (0.5 µg/ml), insulin (5 µg/ml), transferrin (10 µg/ml), epinephrine (0.5 µg/ml), triiodothyronine (6.5 ng/ml), bovine pituitary extract (52 µg/ml), EGF (0.5 ng/ml), all-trans retinoic acid (50 nM, Sigma), penicillin (100 U/ml, Sigma), streptomycin (0.1 mg/ml, Sigma), nonessential amino acids (1x, Sigma), and fatty acid-free bovine serum albumin (3 µg/ml, Sigma). Cells were grown in the BEGM for 1 wk, at which point an air-liquid interface (ALI) culture system was initiated (30). The cells were grown for 2 wk at ALI and fed every other day basolaterally until use in the Ussing chamber (usually 710 days).
Ussing chamber studies. Monolayers of HNE and CFHNE were mounted in modified Ussing chambers (Physiological Instruments, San Diego, CA) with the use of a Ringer bicarbonate solution containing (in mM) 115 NaCl, 2.4 K2HPO4, 0.4 KH2PO4, 1.2 MgCl2, 1.2 CaCl2, 25 NaHCO3, and 10 glucose, unless otherwise indicated. Experiments were carried out at 37°C, and the pH was adjusted to 7.4 by being gassed with 95% O2-5% CO2. After an open-circuit equilibration period of 10 min, the transepithelial PD was recorded, the cells were voltage clamped at 0 mV, and the resulting Isc was continuously recorded. A periodic bipolar voltage pulse monitored resistance calculated using Ohm's law. In acute experiments, drugs were added to the apical or basolateral compartment, as indicated, and the changes in response were recorded. In preincubation experiments, a compound dissolved in 100 µl of media was added to the apical surface of monolayers growing on Snapwells. After a 2-h incubation period at 37°C in a CO2 incubator, the apical media containing compound was removed, and monolayers were washed with BEGM and returned to ALI for the indicated time before being mounted in Ussing chambers.
22Na+ flux. Monolayers of CFHNE were pretreated with either 20 µM INO-4995 or 2-h vehicle control for 1722 h before being tested. Monolayers were mounted in Ussing chambers, and PD and resistance were monitored under open-circuit conditions while ion fluxes were measured as described previously (26). A quantity of 10 µCi of 22Na and 45 µCi of 36Cl were added to either the mucosal or serosal chamber. Aliquots (3 ml) were removed from the downhill chambers for counting at 5-min intervals. After 20 min, 100 µM amiloride was added to the mucosal chamber and aliquots were removed for an additional 20 min. Ion flux was calculated as described.
Patch clamp.
Outside-out patches were configured from 3T3 cells stably expressing rat -ENaC. Patches from 3T3 containing near-silent ENaCs were activated with trypsin (4) before INO exposure. The temperature was
23°C. The 3T3-cell bath solution contained (in mM) 150 Li+-aspartate, 2 MgCl2, 1 CaCl2, and 5 HEPES, titrated to pH 7.30 with LiOH. Calculated osmolarity was 314 mosmol/l. The 3T3-cell pipette solution contained (in mM) 120 Tris-aspartate, 20 NaCl, 3 MgATP, 0.2 Na2GTP, 0.1 CaCl2, 1 EGTA, and 5 HEPES titrated to pH 7.10 with NaOH. Free [Ca2+] was
43 nM, and calculated osmolarity was 295 mosmol/l.
Apical permeabilization and Na+-K+-ATPase activity. Nystatin is a pore-forming antimycotic that enables permeabilization of the cell membrane to monovalent ions: Na+, K+, and Cl (23). Apical nystatin (360 µg/ml) was applied to the solution that bathed the apical compartment at the indicated time. Cytosolic levels of Na+ (25 mM) were achieved in the bathing solutions by replacing a portion of the NaCl with NMDG-C (145 mM). After permeabilization, the increase in Isc is primarily due to Na+ movement driven by the Na+-K+- ATPase that is completely inhibited by basolateral application of 100 µM ouabain. The composition of the buffer was as follows (in mM): 145 NMDG-Cl, 25 Na+, 5 K+, 1.2 Ca2+, 1.2 Mg2+, 25 HCO3, 3.3 H2PO4, 0.8 HPO42, and 10 glucose, pH 7.4.
To create a K+ gradient to evaluate for effects on basolateral K+ channels, the buffers were as follows: basolateral compartment (in mM) 120 K-gluconate, 4 Ca-gluconate, 4 MgSO4, 0.8 K2HPO4, 3.3 KH2PO4, 25 NaHCO3, and 10 glucose; and apical compartment (in mM) 120 Na-gluconate, 4 Ca-gluconate, 4 MgSO4, 0.8 K2HPO4, 3.3 KH2PO4, and 25 glucose.
Blue Dextran fluid transport assay.
Blue Dextran (BD) is a 2,000,000-MW compound that is not absorbed by cells and does not move across tight junctions. BD stock solution was prepared with HEPES-modified Ringer buffer (HMRB) (2 mg BD/ml buffer). INO-4995 was solubilized in HMRB [pH 6.7, containing (in mM): 135 NaCl, 1.2 CaCl2, 1.2 MgCl2, 2.4 K2HPO4, 0.6 KH2PO4, 10 HEPES, and 10 glucose (285 mosmol/l)] containing
1 µM BD. The final concentration of vehicle was 0.1% [1:1, DMSO+DMSO containing 5% (wt/vol) Pluronic F-127]. Two hundred microliters of the BD (1 µM) solution containing vehicle or compound were added apically, and monolayers were incubated (Forma model 3956 set on maximum humidity setting) for 18 h. Control plates with media and impermeable membranes showed no loss of volume over 18 h under these conditions. Basolateral buffer consisted of BEGM (
300 osmol/l). After 18 h, 60 µl of the remaining apical buffer were collected for analysis. A standard concentration curve was obtained from the optical density, at 660 nm, of a serial dilution of 10 µM BD in HMRB in a 96-well plate with the use of a Packard Spectracount. The concentration of BD ([BD]) in the samples was extrapolated from the BD standard curve using Packard I-Smart software. The increase in [BD] from the starting value of 1 µM was taken as the magnitude of volume absorption. Absorption rate was calculated by dividing the
volume by time normalized to monolayer surface area.
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RESULTS |
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Prolonged effects of INO-4995 on CFNHE electrophysiology. We exposed monolayers apically to INO-4995 for 2 h, then washed and returned the cultures to ALI for 22 h before mounting the monolayers in Ussing chambers for testing. At this time, Isc was reduced (Fig. 2A), demonstrating a prolonged duration of action. We repeated this experiment with multiple doses of INO-4995 to generate the dose-response curve depicted in Fig. 2B. INO-4995 dose-dependently inhibited the basal Isc in CFHNE (EC50 = 10 µM; Fig. 2B). In addition to the vehicle controls, additional controls included pretreatment with phosphate PM ester (Pi/PM), or structurally related analogs with fewer phosphates. IP analogs that did not have the membrane-permeating protecting groups were likewise without effect. A series of membrane-permeant IP analogs ranged from having no effect to having similar effects to INO-4995 with potency corresponding to the phosphorylation pattern and the nature of the substitutions at positions 1 and 2. INO-4995 had no apparent deleterious effects on tight junction integrity, as resistance did not decrease (Fig. 2C), even with 500 µM INO-4995. In other experiments, repeated exposures over a period of 48 days likewise did not reveal delayed or cumulative adverse effects on monolayer integrity.
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INO-4995 inhibits ENaC activity in overexpressing cell lines. MDCK cells overexpressing ENaC (45) allowed more direct testing of the effect of INO-4995 on Na+ channel activity. These cells exhibit an unusually high basal Isc that is inhibitable with amiloride and therefore are a good model for CF epithelia. In this model system, as in human airway CF epithelia, ENaC activity is responsible for most of the elevated basal Isc. INO-4995 (5 µM) preincubation inhibited basal Isc measured 24 h later in the ENaC-overexpressing MDCK cells in much the same manner as in the CF airway epithelia (Fig. 3). The majority (91%) of the INO-4995 effect is attributable to the amiloride-inhibitable portion of the current supporting the hypothesis that INO-4995 inhibits ENaC (Fig. 3B).
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22Na+ flux studies. INO-4995 effects on Na+ flux in airway epithelia was tested under open circuit conditions. No effects on electrical parameters or ion flux were seen in initial experiments using up to 50 µM INO-4995 on healthy bovine trachea in keeping with specificity of the compound for CF tissue. In contrast, a 2-h pulsed exposure to 20 µM INO-4995 1722 h before testing resulted in inhibition of basal open circuit PD in human CF nasal epithelia. Corresponding changes in 22Na+ mucosal to serosal and serosal to mucosal flux are shown in Table 1. Both serosal to mucosal and mucosal to serosal amiloride inhibitable 22Na+ flux were absent in INO-4995-treated monolayers, whereas amiloride inhibited serosal-to-mucosal 22Na flux in untreated cells. These results are consistent with amiloride-sensitive fluid absorption being present normally, but being abolished by treatment with INO-4995.
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To further probe whether there might be an effect of INO-4995 on basolateral K+ channel activity, a K+ gradient was applied to monolayers apically permeabilized with nystatin after pretreatment with 10 µM INO-4995 as in the experiments described above. As shown in Fig. 4C, the current driven by the K+ gradient was similar in both INO-4995-treated monolayers and controls.
INO-4995 inhibits fluid absorption. To ascertain the therapeutic relevance of these electrophysiological observations, we evaluated the ability of compounds to inhibit fluid absorption measured directly. The assay consisted of monolayer cultures of polarized CF nasal epithelia exposed to an apical buffer containing test compounds and a known concentration of the nonpermeable BD, using a modification of a protocol described by Matsui and coworkers (30). The observed absorption rates ranged from 4 to 6 µl·cm2·h1, consistent with values reported for CF tissue elsewhere (20). We used amiloride, a blocker of the Na+ channel, ENaC, to assess the ability of this assay to measure relevant changes in fluid secretion. Accordingly, blocking ENaC with amiloride should significantly reduce fluid absorption. As anticipated, amiloride (continuously present for the duration of the 18-h incubation period) inhibits fluid absorption measured by the BD assay (Fig. 5A) in a dose-dependent fashion.
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INO-4995 inhibition of fluid absorption persists for 42 h. The residence time of aerosolized drugs in airways is limited by expulsion, absorption, and hydrolysis. This may contribute to the short duration of action of extracellularly acting agents, such as amiloride or purinergic agonists (38, 54). In contrast, membrane-permeant analogs of IP are not subject to those factors once inside the cell. Therefore, we compared the duration of the effect on fluid absorption following pulsed exposure to compounds. We compared the long-term effect of pulsed exposure to 50 µM INO-4995 (Fig. 5C) to that of amiloride. The data show that INO-4995 exerted prolonged effects on fluid absorption. After a 2-h exposure, INO-4995 continues to affect fluid levels up to 42 h after exposure. In contrast, amiloride effects are short lived, vanishing on removal from surface liquid so that, as expected, no residual effect of amiloride contact is detectable 42 h after exposure (Fig. 5C). On the basis of these results, INO-4995 is likely to be a much more effective therapeutic than amiloride due to prolonged duration of action.
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DISCUSSION |
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The target of INO-4995, resulting in inhibition of basal Isc, is located at the apical membrane. Apical membrane permeabilization of monolayers eliminated the Isc differential between INO-4995 pretreated monolayers and control. There was no inhibition of basolateral Na+-K+-ATPase activity after INO-4995 treatment. Likewise, K+ currents in monolayers apically permeabilized were unchanged in the INO-4995-treated monolayers in the presence of a K+ gradient (Fig. 4C). These data indicate that INO-4995 inhibits Na+ absorption through an apical, amiloride-sensitive channel, probably ENaC. This is supported by the following data. First, the elevated basal Isc characteristic of CF tissue that is inhibited by INO-4995 is primarily due to absorption of Na+ via ENaC. Second, INO-4995 inhibited the amiloride-sensitive Isc in MDCK cells overexpressing ENaC where the elevated basal Isc is attributable to ENaC. Third, INO-4995 inhibited current recorded via 2-electrode voltage clamp in ENaC transfected Xenopus oocytes (S. Gabriel, unpublished observations). Fourth, INO-4995 inhibited 22Na+ flux in CF human epithelia. Finally, INO-4995 inhibited ENaC currents in outside-out patches from 3T3 cells overexpressing rat -ENaC.
INO-4995 does not act in the same manner as amiloride. First, while amiloride acts extracellularly, INO-4995 acts intracellularly. Second, amiloride action is short-lived and dependent on the continuous presence of the compound, whereas INO-4995 action is prolonged. Third, amiloride potency in normal HNE is similar to that in CFHNE. In contrast, INO-4995 is much more potent in CFHNE than in epithelia from non-CF donors.
INO-4995 reduced the rate of amiloride-inhibitable fluid reabsorption in CF epithelia using a BD assay. There was a component of the fluid absorption rate that was neither inhibited by amiloride nor INO-4995. This may reflect evaporative loss or an uncharacterized conductance. However, the maximum change in the rate of fluid flux with amiloride in the BD assay (2.5 µl·cm2·h1) is very close to the amiloride-inhibitable flux calculated from the 22Na flux studies (3.3 µl·cm2·h1) assuming isotonicity of transported fluid. Tarran and associates (47) have shown that fluid absorption rates slow when the height of the ASL falls <20 µm. However, under the conditions of the current study, the height of the surface fluid remains well above this level during the entire experiment. Further studies are planned which could determine whether there are differential effects of INO-4995 with varying fluid levels. It should be noted that an amiloride-sensitive fluid absorption of 3 µl·cm2·h1 is similar to that reported by others (20) and, if unopposed, would shrink the depth of ASL by 0.5 µm/min. Therefore, if inhibiting amiloride-sensitive Na+ absorption unmasks an opposing secretory process, then ASL depth could increase by as much as 0.5 µm/min, causing ASL to double in depth approximately every 10 to 20 min.
The key to the therapeutic potential of INO-4995 is its prolonged effect that may be due to slow metabolism of the active form, INO-4913. Ins(3,4,5,6)P4 levels can remain elevated for hours (50) and are controlled by the reversible Ins(3,4,5,6)P4/InsP5 1-kinase/1-phosphatase (15) that phosphorylates Ins(3,4,5,6)P4 on the 1 position, generating InsP5. The deesterified product of the prodrug INO-4995, INO-4913 (Fig. 1), cannot be directly metabolized via this pathway due to the ether-linked octyl group at the 1 position, contributing to its extended duration of its action.
We considered the possibility that INO-4995 could be acting through modulation of serine protease activity, a recently characterized path that modulates ENaC open probability (7). However, INO-4995 appears to act in a serine protease-independent manner because monolayers pretreated with 10 µM INO-4995 still exhibited reduced basal Isc relative to control after the addition of trypsin (data not shown). Moreover, the addition of aprotinin, a serine protease inhibitor, further reduced basal Isc in INO-4995 pretreated monolayers in an additive fashion, indicating that the two agents work through separate but complementary mechanisms.
Phosphatidylinositol (PI)3-kinase pathways can regulate ENaC surface expression in renal epithelia. For instance, Nedd4-2 regulates ENaC expression (31, 43, 44) and is downstream of hormone (aldosterone or insulin) triggered serum and glucocorticoid-regulated kinase (Sgk-1) and PI3-kinase signaling (1, 6, 11). Certain IPs interfere with PI3-kinase signaling pathways (12) and may therefore reverse PI3-kinase-mediated Sgk-1 upregulation of ENaC via Nedd4-2.
INO-4995 may directly modulate ENaC. Herein we have shown that INO-4995 inhibited ENaC open probability in excised outside-out patches. Although the concentration of INO-4995 in the bath was 20 µM, the compound would presumably have to cross the patch membrane and be deesterified to generate the active compound, INO-4913, at the site of action. Therefore, a much more detailed study is underway to determine the concentration of the relevant compound and pinpoint the site of action. INO-4995 may indirectly modulate ENaC through actions on Cl channels. For instance, INO-4995 is an analog of Ins(3,4,5,6)P4, which modulates Ca2+-activated Cl channels in multiple systems (5, 19, 42, 5052). We have shown that an analog of INO-4995 opens Cl channels in whole cell patch-clamp configuration (9), whereas a deesterified form of INO-4995, INO-4913 opens Cl channels in a dialyzed cell preparation (10). In addition, repeated exposure to low doses of INO-4995 enhances the Isc response to ATP, suggesting an increase in Ca2+-activated Cl channel activity (33). Others have shown that Ca2+-activated Cl channels as well as CFTR activity can modulate Na+ flux via electrochemical coupling (16, 24, 25, 34). A recent study (53) suggests that both ENaC activity and expression levels are modulated by Cl channel-mediated changes in intracellular Cl levels. However, it is unclear how that mechanism could account for the inhibition of ENaC measured in excised patches described herein.
We have demonstrated that the IP analog, INO-4995, reverses physiological processes associated with pathological function in CF tissue, Na+, and fluid absorption. Thus INO-4995 is a potential treatment for the underlying defect in fluid secretion in CF linked to morbidity and mortality. Our findings support preclinical development of INO-4995 for the treatment of CF and represent the first demonstration of the therapeutic significance of a derivative of an IP signaling molecule. In addition to a potential breakthrough in CF research, our results have much wider implications for the design of pharmaceuticals targeting intracellular IP and phosphoinositide signaling cascades.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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