Channel-forming peptide modulates transepithelial electrical conductance and solute permeability

James R. Broughman,1 Robert M. Brandt,2 Christy Hastings,1 Takeo Iwamoto,2 John M. Tomich,2 and Bruce D. Schultz1

1Anatomy and Physiology and 2Biochemistry, Kansas State University, Manhattan, Kansas 66506

Submitted 16 September 2002 ; accepted in final form 14 January 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
NC-1059, a synthetic channel-forming peptide, transiently increases transepithelial electrical conductance (gTE) and ion transport (as indicated by short-circuit current) across Madin-Darby canine kidney (MDCK) cell monolayers in a time- and concentration-dependent manner when apically exposed. gTE increases from <2 to >40 mS/cm2 over the low to middle micromolar range. Dextran polymer (9.5 but not 77 kDa) permeates the monolayer following apical NC-1059 exposure, suggesting that modulation of the paracellular pathway accounts for changes in gTE. However, concomitant alterations in junctional protein localization (zonula occludens-1, occludin) and cellular morphology are not observed. Effects of NC-1059 on MDCK gTE occur in nominally Cl- and Na+-free apical media, indicating that permeation by these ions is not required for effects on gTE, although two-electrode voltage-clamp assays with Xenopus oocytes suggest that both Cl and Na+ permeate NC-1059 channels with a modest Cl permselectivity (PCl:PNa = 1.3). MDCK monolayers can be exposed to multiple NC-1059 treatments over days to weeks without diminution of response, alteration in the time course, or loss of responsiveness to physiological and pharmacological secretagogues. Together, these results suggest that NC-1059 represents a valuable tool to investigate tight junction regulation and may be a lead compound for therapeutic interventions.

transepithelial resistance; cystic fibrosis; tight junction; epithelial barrier; amphipathic {alpha}-helix


A SYNTHETIC PEPTIDE BASED on the second transmembrane segment of the glycine receptor (M2GlyR) has been shown to form an anion-selective pathway in epithelial cells that allows for electrolyte secretion when applied to the apical surface (26). This activity represents a potential therapeutic modality for the treatment of cystic fibrosis (CF) and provides impetus for ongoing development. M2GlyR has been modified to increase aqueous solubility while maintaining channel-forming ability and anion selectivity (NK4-M2GlyR and CK4-M2GlyR; Refs. 2, 33). Several derivatives of this sequence have been shown to be effective at increasing short-circuit current (Isc), an indicator of net anion secretion (2, 3, 31). Studies employing modified peptides suggest that the NH2-terminal half of the transmembrane segment contributes to intramembrane helical bundle formation, whereas the COOH-terminal half of the segment is responsible for aggregation in aqueous solution (3). Thus a peptide has been designed and synthesized (KKKK-PARVGLGITTVLVTTIGLGVRAP; designated NC-1059) in which the transmembrane portion of the molecule is a palindrome of the bundle-forming, nonaggregating, NH4-terminal portion around the central leucine. NC-1059 elicits an increase in Isc at low micromolar concentrations and remains monomeric in aqueous solution (3). NK4-M2GlyR requires a fourfold higher concentration than NC-1059 to support anion flux [EC50 = 208 ± 6 µM (2) vs. 50 ± 3 µM (3)]. Thus NC-1059 appears to exhibit all targeted attributes for a CF therapeutic (monomeric, soluble, low cytotoxicity). Additionally, NC-1059 causes an increase in the transepithelial electrical conductance (gTE) across Madin-Darby canine kidney (MDCK) monolayers, which might suggest an effect on the paracellular pathway and specifically on tight junctions. This latter effect, which has not been reported previously for any synthetic peptide, appears to preclude the palindromic peptide from further consideration as a CF therapeutic. However, NC-1059 represents a possible tool to study or manipulate the epithelial barrier.

Tight junctions are complex, highly regulated, dynamic structures that are a barrier to movement of solutes between apical and basolateral compartments and form a "fence" that maintains cell membrane polarity (4, 37). Regulated openings of junctions occur in a variety of situations such as sperm maturation (18), extravasation of lymphocytes (8), and nutrient uptake in the intestine (23). Pathology associated with aberrant function and dysregulation of tight junctions includes cancer metastases (16), autoimmune dysfunction (20), and inflammatory bowel disease (12), to name just a few. Tight junctions are targets of bacterial toxins such as the Vibrio cholerae zonula occludens toxin (ZOT) and Clostridium difficile toxins TcdA and TcdB (36) in pathological, experimental, and perhaps therapeutic situations (9, 10). A mammalian homolog of ZOT has been identified and may be a primary physiological regulator of tight junctions in intestinal tissues (36). Additionally, cytokines and a number of second messengers are known to be involved in the modulation of tight junctions (8, 9, 14, 34, 37), although the mechanisms by which these processes occur remain to be elucidated.

A variety of treatments, including Ca2+ chelation, surfactants, fatty acids, and cationic polymers, have been used to experimentally modulate the paracellular pathway (17, 37). However, when applied in vivo, side effects of the chemical treatments used to modulate gTE can include hypersensitivity, asthma, anaphylaxis, and the sloughing of epithelial cells (9, 17, 37). Surfactants and detergents can cause cell lysis and sloughing, whereas Ca2+ chelators can cause cytoskeletal rearrangements and interfere with calcium signaling pathways (37). An additional drawback to these treatments is that, in general, there is little separation between the effective concentration and the cytotoxic concentration (37). Alternatively, ZOT, which lacks many undesirable side effects, has been used to modulate the epithelial barrier in an experimental therapeutic setting (10). The results are encouraging and provide a "proof of concept" indicating that intestinal tight junctions can be modulated to allow for the absorption of high-molecular-weight therapeutic compounds without apparent deleterious side effects (10). Other studies (5) have suggested that gene therapy might be augmented by transiently reducing epithelial tight junction integrity as well. Thus there is a great need to identify safe and efficient means by which to modulate the epithelial barrier function at the tight junction in a wider variety of epithelia.

The goal of the present study is to define the relationship between the apical exposure of epithelial cells to NC-1059, a channel-forming peptide, to changes in barrier function as typified by the increase in gTE and transepithelial solute permeability. Results suggest that the peptide provides a route for ion permeation across the cell membrane and modulates the paracellular pathway over a similar concentration range, although the time course of the two responses is dramatically different.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Peptide synthesis. All peptides were synthesized by solid phase synthesis using 9-fluorenylmethoxycarbonyl chemistry as described in detail previously (31). Peptides were purified and characterized by reverse-phase HPLC and matrix-assisted laser desorption time-of-flight mass spectroscopy (MALDI-TOF).

Cell culture. MDCK cells were provided by Dr. Lawrence Sullivan (University of Kansas Medical Center, Kansas City, KS) and were maintained in culture as described previously (2). Briefly, the culture medium was a 1:1 mixture of DMEM and Ham's F-12 (GIBCO BRL, Rockville, MD) supplemented with 5% heat-inactivated FBS (BioWhittaker, Walkersville, MD) and 1% penicillin and streptomycin (GIBCO BRL). Cells were grown in plastic 25-cm2 culture flasks (Cellstar, Frickenhausen, Germany) in a humidified environment with 5% CO2 at 37°C. Confluent cultures were dissociated for subculture with PBS containing 2.6 mM EDTA and 0.25% trypsin. For permeation and flux experiments, cells were seeded on 1.13-cm2 permeable supports (Snapwell; Costar, Cambridge, MA) at a density of ~1 x 106 cells/well and incubated in DMEM/F-12 supplemented with FBS and antibiotics (refreshed every other day) for 2–3 wk before cells were mounted in modified Ussing flux chambers for evaluation.

Electrical measurements. Transepithelial ion transport was evaluated in modified Ussing chambers (Model DCV9; Navicyte, San Diego, CA). For typical electrical measurements of ion flux, cells were bathed in symmetrical Ringer solution (composition in mM: 120 NaCl, 25 NaHCO3, 3.3 KH2PO4, 0.8 K2HPO4, 1.2 MgCl2, 1.2 CaCl2; 290 ± 2 mosmol/kgH2O). The diffusion chambers were maintained at 37°C and continuously bubbled with 5% CO2-95% O2 to maintain pH, provide aeration, and mix the fluid in the chambers. The transepithelial potential was clamped to zero, and Isc was measured continuously with a voltage-clamp apparatus (Model 558C; Department of Bioengineering, University of Iowa, Iowa City, IA). Data were acquired at 1 Hz with a Macintosh computer (Apple Computer, Cuppertino, CA) using Aqknowledge software (v. 3.2.6; BIOPAC Systems, Santa Barbara, CA) with an MP100A-CE interface. gTE was determined by exposing the epithelia to a 5-s, 1-mV, bipolar pulse at 100-s intervals. The recorded current deflections were used with Ohm's law to calculate gTE (gTE = {Delta}I/{Delta}V).

Alternative apical bathing solutions that allowed for the imposition of defined transepithelial ion gradients were employed for one set of experiments. Three solutions of virtually identical osmolality (280–290 mosmol/kgH2O) and total electrolyte strength to normal Ringer solution were formulated: nominally Na+-free [in mM: 120 N-methyl-D-glucamine (NMDG)·Cl, 25 choline-HCO3, 3.3 KH2PO4, 0.8 K2HPO4, 1.2 CaCl2, 1.2 MgCl2], nominally Cl-free (in mM: 120 Na-gluconate, 25 NaHCO3, 3.3 KH2PO4, 0.8 K2HPO4, 1.2 CaSO4, 1.2 MgSO4, 2.8 CaCl2), and nominally NaCl-free (in mM: 120 NMDG-gluconate, 25 choline-HCO3, 3.3 KH2PO4, 0.8 K2HPO4, 1.2 CaSO4, 1.2 MgSO4, 2.8 CaCl2). CaCl2 was added to the gluconate-containing solutions to maintain the free Ca2+ concentration and to ensure adequate Cl for proper electrode function.

Xenopus oocyte isolation. Oocyte isolation was performed as described previously with minor modifications (15, 28, 30). Briefly, sexually mature, human chorionic gonadotropin-treated Xenopus laevis were purchased (Xenopus 1, Ann Arbor, MI) and individually maintained in aquaria in an Association for Assessment and Accreditation of Laboratory Animal Care-accredited facility. Oocyte isolation was accomplished by using Institutional Animal Care and Use Committee-approved protocols in which Xenopus were anesthetized by exposure to MS-222 (Sigma, St. Louis, MO) and a laparoscopic approach was employed to isolate and remove the ovary. Oocytes were separated from follicular cells by incubation in nominally Ca2+-free ND-96 (in mM: 96 NaCl, 1 KCl, 1 MgCl2, 5 HEPES, pH 7.5) including 0.7 mg/ml collagenase (GIBCO BRL) and 0.1 mg/ml trypsin inhibitor (Sigma) on a low-speed rocker at room temperature for 35–60 min. Oocytes were rinsed five times and incubated in K2HPO4 (100 mM; pH 6.5) with BSA (0.1% wt/vol; Sigma) for 1 h with gentle agitation at 15-min intervals. Oocytes were then transferred to and maintained in modified Barth's solution [in mM: 88 NaCl, 2.4 NaHCO3, 1 KCl, 0.82 MgSO4, 0.41 CaCl2, 0.3 Ca(NO3)2, 10 HEPES, pH 7.5] at 18–20°C until current recordings were made 1 to 5 days later.

Membrane conductance and permselectivity. The two-electrode voltage-clamp technique was employed. Oocytes were impaled with two 3-M KCl-filled electrodes having resistances of 0.5–2 M{Omega}. The electrodes were connected to a GeneClamp 500 current-voltage clamp amplifier (Axon Instruments, Foster City, CA) via Ag-AgCl pellet electrodes and referenced to a Ag-AgCl pellet that communicated to the bath via an agarose bridge (3% agarose in 1 M KCl). The voltage clamp was controlled by an analog-digital interface (Digidata 1200b) using a Pentium-based computer running pClamp software (version 9.0; Axon Instruments) for command potential and current and voltage recording. Two voltage-pulse protocols were employed. In the first, membrane potential (Vm) was held at –30 mV (approximately the resting Vm) and pulsed to –90 mV for 1,000 ms, returned to –30 mV for 1,000 ms, and then pulsed to 0 mV for 1,000 ms. This pulse protocol was repeated at 4,128-ms intervals throughout the experimental period to verify that stable conductance levels were achieved with each change of bath solution. Current-voltage (I-V) relationships were generated at the end of each treatment period (baseline, peptide-exposed, ion-substituted) with a repeating three-step protocol. Vm was held at –30 mV for 500 ms, pulsed to one of nine voltages (–100 to +60 mV in 20-mV increments) for 1,000 ms, and returned to –30 mV for 500 ms. The average voltage and current during the final 500 ms of each voltage pulse was used to construct each I-V relationship.

Data were recorded in solutions of four ionic compositions: ND-96 (in mM: 96 NaCl, 1 KCl, 1 MgCl2, 1.8 CaCl2, 5 HEPES), reduced Na+ and Cl (in mM: 173 mannitol, 9.6 NaCl, 1 KCl, 1 MgCl2, 1.8 CaCl2, 5 HEPES), reduced Cl (in mM: 92.3 Na-gluconate, 3.7 NaCl, 1 KCl, 1 MgCl2, 1.8 CaCl2, 5 HEPES), and reduced Na+ (in mM: 86.4 NMDG·Cl, 9.6 NaCl, 1 KCl, 1 MgCl2, 1.8 CaCl2, 5 HEPES). pH was adjusted to 7.5 for all solutions. The final osmolality of all solutions was between 185 and 200 mosmol/kgH2O. I-V relationships were initially recorded in ND-96 in the absence of any synthetic peptide. Peptide (8 µl, 5 mM in H2O) was then added to the 400-µl bath and mixed to attain a final concentration of 100 µM. After stable parameters were attained (<5 min), an I-V relationship was again recorded. Subsequently, the bath composition was changed to an alternative ion composition by adding 200 µl of the new solution, mixing, and removing 200 µl of the bathing medium 15 times (>99.7% bath replacement). Peptide was again added to the bathing medium (100 µM final concentration), and the stability of membrane conductance was verified before an I-V relationship was recorded. The bathing medium was repeatedly changed with this technique. In every case, however, recordings were made in ND-96 before (and typically after) recording in an alternative ionic composition to verify reversibility of bath composition-induced changes and to allow for comparisons to temporally close controls. Visual inspection suggested that a linear I-V relationship was present, as expected. Thus linear regression (Sigmaplot v. 2000 for Windows, SPSS, Chicago, IL; and Excel, v. 9.0.38, Microsoft, Redmond, WA) was employed to determine the slope conductance and reversal potential (Vrev) in each condition. I-V relationships were mathematically adjusted for junction potentials by using the appropriate pClamp module. Permselectivity (P) for Cl vs. Na+ was estimated by using Eq. 1, which is derived from the Goldman-Hodgkin-Katz constant field equation (13)

(1)
This analysis is based on changes ({Delta}) in Vrev that accompany a change in bath ionic composition with the underlying assumption that, in the presence of peptide, membrane conductance could be attributed to the peptide. The analysis further assumes that, since the concentration of other ions (e.g., K+, Ca2+, Mg2+) was relatively small and unchanged, overall permeation by these ions would minimally contribute to changes in Vrev. e, F, R, and T have their conventional definitions. Subscript 1 indicates the ion activity in ND-96, whereas subscript 2 denotes the activity in reduced-NaCl ND-96.

FITC-dextran permeability assay. Epithelial permeability to uncharged solutes of various sizes was assessed with monolayers grown on Snapwell tissue culture inserts as described in Cell culture. Confluent monolayers were washed once with Ringer solution and placed in one of three treatments containing FITC-conjugated dextran (Sigma) in the apical compartment: 1) Ringer solution in the apical and basolateral compartments; 2) Ringer solution in the apical and basolateral compartments with NC-1059 (100 µM) in the apical solution; or 3) EDTA (3 mM) in Ringer solution that was diluted 1:1 with distilled water in both the apical and basolateral compartments. Monolayers were incubated at 37°C for 60 min, and the solution in the basolateral well was sampled to quantify fluorescently labeled dextran. Monolayers were then washed with Ringer solution to remove peptide, EDTA, and dextran, placed in tissue culture medium, and returned to the incubator for 2 days before the assay was conducted again.

Confocal microscopy. Immunoreactivity to antibodies raised against tight junction-associated proteins was assessed by confocal microscopy (Zeiss, Thornwood, NY). Samples for visualization were prepared from monolayers used in electrophysiological studies. After being removed from Ussing chambers, monolayers were washed in Ringer solution and fixed overnight in 10% neutral buffered formalin. Monolayers were washed three times in PBS, permeabilized with 0.1% Triton X-100 in PBS, blocked with goat serum, and then exposed to primary antibody in a 1:500 dilution (rat anti-zonula occludens-1, catalog no. MAB120; Chemicon, Temecula, CA, or rabbit anti-occludin, catalog no. 71-1500; Zymed, San Francisco, CA) for 1 h at room temperature. After being washed three times in PBS, FITC-conjugated goat anti-rat (catalog no. AP136F; Chemicon) or goat anti-rabbit (catalog no. Fl-1000; Vector Laboratories, Burlingame, CA) secondary antibodies were employed (1:1,000 dilution) with exposure occurring for 1 h at room temperature. TRITC-labeled phalloidin (0.1 mg/ml in methanol; Sigma) used for F-actin localization was applied concurrently with the secondary antibody. A KrAr laser was used to excite the fluorophores. Filter sets used for fluorescein were band pass (BP) 485 ± 20 nm for excitation and BP 515 ± 540 nm for emission, and for rhodamine the sets used were BP 530 ± 585 nm for excitation and long pass (LP) 590 nm for emission.

Chemicals and stock solutions. 1-EBIO (Acros; Fisher Scientific, Pittsburgh, PA) was prepared as a 1 M stock solution in DMSO. Forskolin (Coleus forskohlii) was purchased from Calbiochem (La Jolla, CA) and prepared as a 10 mM stock in ethanol. All other chemicals were purchased from Sigma and were of reagent grade unless otherwise noted. Unless otherwise stated, synthetic peptide was suspended in water at 5 mM just before experimental addition.

Data analysis. All results are presented as means ± SE. Fitting of user-defined functions to data sets was conducted with Sigmaplot. The difference between treatment groups was analyzed by using Student's t-test (Microsoft Excel 2002). The probability of making a type I error <0.05 is considered statistically significant. The reported value of n is the number of independent observations.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
NC-1059 mediates ion transport in a concentration-dependent manner. NC-1059 causes a concentration-dependent increase in Isc (indicative of anion secretion or cation absorption) and gTE across MDCK epithelial monolayers (2). Thus experiments have been conducted to determine if the concentration dependence is similar for these two outcomes. Epithelia were pretreated with 1-EBIO to fully activate basolateral potassium channels in all preparations and thus maximize the electrochemical driving force for ion transport (2). As previously reported, 1-EBIO has no effect on either Isc or gTE, suggesting that under basal conditions the basolateral membrane of MDCK cells is neither rate limiting for anion secretion nor the primary determinant of transepithelial resistance (Fig. 1, A and B). Apical exposure to NC-1059 (100 µM) results in a rapid increase in Isc that reaches a peak value before declining toward zero (Fig. 1A) with corresponding changes in gTE (Fig. 1B). Basal gTE is low (<1 mS/cm2), and exposure to NC-1059 results in an increase in gTE with a slower onset of effect than the increase in Isc. These results suggest that the conductive pathway that accounts for ion transport does not directly or fully account for the increase in gTE. However, the increase in gTE may contribute to the return of Isc toward zero by depolarization of the epithelial cells that reduces secondary active ion flux. There is also the possibility that, at extremely high gTE, any electrode offset or junction potential could affect Isc. Data similar to that presented in Fig. 1A, in which the change in Isc is plotted as a function of peptide concentration, have been reported previously (3). The fit of a modified Hill equation to the data revealed a value for K1/2 (50 µM) that is fourfold lower than that reported for similar peptides (e.g., NK4-M2GlyR = 208 ± 6 µM), although the predicted maximal short-circuit current (25.0 µA/cm2) is indistinguishable (24.3 ± 0.5; Ref. 2). These results show that NC-1059 exhibits greater biological availability or efficacy than NK4-M2GlyR while maintaining channel-forming ability, a stated goal for the design of a CF therapeutic. Additionally, substantial increases in gTE are observed. Whereas the latter effect is not a targeted outcome, it is immediately obvious that NC-1059 provides unique research and therapeutic opportunities. Characterization of this effect provides the basis for the remainder of this report.



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Fig. 1. NC-1059 causes a concentration- and time-dependent increase in transepithelial ion transport (short-circuit current; Isc) and conductance (gTE). A: representative experiment showing effect of NC-1059 on Isc and gTE across a Madin-Darby canine kidney (MDCK) monolayer. Solid line represents the current magnitude at 0 mV with an upward change indicative of net anion secretion or cation absorption. Periodic deflections result from the imposition of a 1-mV bipolar pulse that is used to determine gTE. B: values for gTE derived by using Ohm's law for each voltage-induced current deflection in A. Dashed lines represent either zero current (A) or zero conductance (B). C: time-dependent increase in conductance across MDCK monolayers at increasing NC-1059 concentrations [20 µM ({blacksquare}), 30 µM ({bullet}), 40 µM ({blacktriangleup}), 50 M ({blacktriangledown}), 60 µM ({blacklozenge})]. Data are means ± SE for 5–15 observations. Solid lines represent the best fit of Eq. 2 (see text for fitted parameters) to each data set. D: derived maximal change in gTE plotted as a function of peptide concentration. Data are means ± SE derived by the fit of Eq. 2 to the data set for each NC-1059 concentration.

 
NC-1059 causes concentration-dependent increase in MDCK gTE. Experiments have been conducted to determine the concentration dependence and time course of NC-1059-induced changes in gTE. Data from Fig. 1B and 3–10 additional experiments at each concentration are summarized in Fig. 1, C and D. Results clearly show that gTE increases to a plateau value over the duration examined and that the peptide-induced increase in gTE is concentration dependent. To predict the maximal change in gTE and the time course of this change, data in Fig. 1D were fitted by a logistic equation.

(2)
gTE0 is the initial gTE, and gTEmax represents the maximal change in gTE. t0 represents the time to reach gTEmax/2, and b is inversely proportional to the rate of rise at t0. For the analysis, gTE0 was constrained to be positive, because negative numbers are, in this case, nonsensical. Derived values of t0 varied over a narrow range (12.4 ± 1.5 min at 60 µM to 15.3 ± 0.6 min at 30 µM), with no distinct concentration dependence observed. The rate of increase in gTE (b) exhibits concentration dependence, with a maximal rate derived for exposure to 60 µM NC-1059 (5.1 ± 0.6/min). Likewise, the gTEmax was also concentration dependent, with a maximum value of 33.5 ± 2.2 mS/cm2, which is approaching a practical limit for the assay system in that this conductance is one-third to one-half the electrical conductance of a culture insert in the absence of cells (70–100 mS/cm2). Values of gTEmax derived from the mathematical fits to the data are plotted as a function of peptide concentration in Fig. 1D. No indication of saturation is observed over the concentration range that could be tested. Whether a maximal gTE is reached cannot be determined effectively, because the observed conductance with 60 µM is approaching the maximal observable conductance for the recording system.

NC-1059-induced changes in gTE require apical exposure. Experiments were conducted to test for the relative efficacy of apical and basolateral NC-1059 on changes in ion transport and barrier function. Results presented in Fig. 2 demonstrate that apical exposure is required to observe a significant effect of the peptide on these parameters. Results from a typical experiment are presented in Fig. 2, AD. When apically exposed to NC-1059 (300 µM), Isc rapidly increases to a peak value and then declines (Fig. 2, A and B), whereas the increase in gTE is delayed (Fig. 2, C and D, consistent with Fig. 1). Exposure of the basolateral membrane to NC-1059 produces no obvious effect, regardless of the order of exposure. Results from these and five additional monolayer pairs are summarized in Fig. 2E. On a pairwise basis, effects were never observed with basolateral exposure and were always observed with apical exposure. These results might suggest that NC-1059 interacts with a cellular component that is accessible only from the apical aspect of the monolayer, although additional experiments would be required to fully test this hypothesis.



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Fig. 2. MDCK monolayers respond to apical, but not basolateral, NC-1059 with a rapid increase in Isc followed by an increase in gTE that remains elevated above baseline for the duration of exposure. AD: results from typical pairs (AB and CD) of monolayers that are exposed to apical and/or basolateral NC-1059 (300 µM) as indicated. E: summarized results from 6 monolayer pairs. 1-EBIO (300 µM) was present throughout all experiments. Dashed lines in AD represent either zero current or zero conductance. Ap, apical; Bl, basolateral.

 
NC-1059-induced changes in gTE are readily reversible. Data presented in Fig. 3 show that the peptide-induced increase in Isc and gTE can reverse over time either with or without washout of the peptide. In the presence of 1-EBIO, 60 µM NC-1059 causes a typical rise in Isc (Fig. 3A) and gTE (Fig. 3B). Following 30 min of exposure, the solution bathing the apical aspect of the epithelium was replaced with peptide-free Ringer solution and recording was continued for 5 h. Results show that the Isc and gTE return toward, but do not return to, pretreatment values during the duration of the recording. In this paradigm, monolayers respond to the subsequent addition of forskolin with an increase in Isc (data not shown), demonstrating that epithelia remain viable and responsive to cAMP-mediated stimulation following peptide exposure.



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Fig. 3. MDCK monolayers respond to treatment with NC-1059 with a rapid increase in Isc (A and C) followed by an increase in gTE that remains above baseline for the duration of exposure (B and D). An MDCK monolayer was exposed to NC-1059 (60 µM) for 30 min as indicated, and the solution bathing the apical surface was replaced with fresh Ringer solution. B: gTE derived for all time points represented in A. C: a separate MDCK monolayer was exposed to 30 µM NC-1059 with no replacement of the apical solution. After reaching a maximum, Isc returned toward the baseline value. The monolayer responded to a second addition of NC-1059, which increased the concentration to 100 µM as indicated, with the expected increase in Isc. D: gTE derived for all time points represented in C. Results are typical of 3–6 separate experiments. Dashed lines represent either zero current or zero conductance.

 
A separate MDCK monolayer was sequentially exposed to two concentrations of NC-1059 (35 and 100 µM) with the recorded Isc and derived gTE presented in Fig. 3, C and D, respectively. Results clearly show that a submaximal increase in gTE is achieved and maintained for over 3 h in the presence of 35 µM NC-1059. Such prolonged exposure is apparently without deleterious effects on the epithelium, because no evidence of epithelial deterioration is observed and responsiveness is maintained. Subsequent exposure to greater concentrations of NC-1059 (100 µM final) further increases gTE and Isc to a value expected for this concentration (17 mS/cm2 and 32 µA/cm2). It should be noted that, regardless of whether the peptide is removed from the bath, some reversal in both Isc and gTE is observed.

Experiments designed to determine whether the NC-1059 activity in solution declines over time were conducted, although it was previously reported that NC-1059 does not aggregate in solution, as was seen with related peptides (3). Results from a typical experiment are presented in Fig. 4. Paired monolayers were mounted in Ussing chambers, with some being immediately exposed to NC-1059. NC-1059 (60 and 100 µM) elicited expected increases in Isc and gTE that reversed over time. Sixty percent of the apical solution was then transferred from the apical side of a treated monolayer to an untreated monolayer, as indicated by the arrows (Fig. 4, A to C and E to G). A previously untreated monolayer was exposed to 60 µM of freshly dissolved NC-1059 at the same time (Fig. 4, D and H). The results clearly demonstrate that active NC-1059 continued to be present in the apical solution even as the effects on Isc and gTE were reversing. The possibility remained that peptide activity was slowly decreasing such that the response declined incrementally as activity diminished. This possibility was excluded by experiments in which a peptide-induced response was generated and the apical solution was partially replaced with Ringer solution containing freshly dissolved peptide. No increment in either Isc or gTE was observed (n = 3). Together, these results suggest that the effect of NC-1059 on MDCK electrical parameters is transient.



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Fig. 4. Transient nature of NC-1059 on Isc and gTE occurs in the presence of active peptide. A and E: an MDCK monolayer was exposed to NC-1059 (100 µM) as indicated. The arrow indicates the time at which the peptide concentration was reduced by 60% removal of the apical solution, which was then used for exposure of another monolayer (C and G). Apical solution was replaced with Ringer solution. B and F: paired time control for A and E showing the effect of 60 µM NC-1059. C and G: an MDCK monolayer was exposed to NC-1059 (60 µM) as indicated. The arrow indicates that apical solution containing peptide that had been in contact with an epithelial monolayer for >75 min (A and E) replaced an equal volume of apical solution. D and H: paired time control for C and G showing the effect of 60 µM freshly dissolved NC-1059. Results are typical of 3 separate experiments. Dashed lines represent either zero current or zero conductance.

 
Additional experiments have been conducted to test for complete reversibility of peptide-induced changes in gTE. Monolayers were exposed to peptide (60 µM) in an Ussing chamber for ~20 min to document increases in gTE, recovered, and returned to the cell culture incubator for 2–5 days with the apical and basolateral media refreshed daily before subsequent assessment of basal gTE and responsiveness to peptide. Results presented in Fig. 5, AC, show the time course of changes in gTE for three typical monolayers that were repeatedly exposed to NC-1059 at 2-, 3-, and 4-day intervals, respectively. Solid arrows indicate the effect of NC-1059, whereas the dashed arrows indicate the return to pretreatment values that is observed before a subsequent exposure. In all cases gTE is <2 mS/cm2 at baseline, increases to >15 mS/cm2 with peptide exposure, and returns to <2 mS/cm2 before the subsequent assessment. Regardless of the duration between peptide exposures (2, 3, or 4 days), gTE of previously treated monolayers returns to pretreatment values and is indistinguishable from that of untreated monolayers (not shown). Some monolayers have been exposed to peptide as many as eight times over a 16-day period with no change in pretreatment gTE and no diminution of responsiveness. Data from four to eight monolayers at each time point are summarized in Fig. 5D. Results demonstrate that gTE after as little as 2 days of incubation in peptide-free medium is indistinguishable from untreated monolayers (0.6 ± 0.1 vs. 0.5 ± 0.1 mS/cm2, respectively). Neither the pretreatment gTE nor the posttreatment gTE differs (P > 0.2) between monolayers that are incubated for 2, 3, 4, or 5 days following peptide exposure. Together, results presented in Figs. 3, 4, and 5 demonstrate that NC-1059-induced changes in gTE are fully reversible and that the responses are readily repeatable.



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Fig. 5. Effect of NC-1059 on gTE is both transient and repeatable. gTE was determined before and after MDCK cell monolayers were exposed to 60 µM NC-1059 on a treatment day. After each exposure, monolayers were removed from the Ussing chamber and returned to the incubator for 2, 3, 4, or 5 days before again being evaluated and exposed to NC-1059. AC: representative data from individual monolayers exposed to NC-1059 at 2-, 3-, and 4-day intervals, respectively. Solid circles represent derived values for gTE. Solid arrows indicate the change in gTE caused by 20-min exposure to NC-1059 (60 µM). Dashed arrows indicate the return of gTE to pretreatment values. No inferences are made regarding the kinetics of the response. D: summary data from 4–8 monolayers that are allowed to recover for 2, 3, 4 or 5 days, as indicated. No significant differences between treatment intervals are observed for preexposure (Pre) and for postexposure (Post) gTE values.

 
NC-1059-induced changes in gTE occur in the absence of small monovalent ions. Experiments have been conducted to test for effects of changing the primary anion and/or cation in the solution bathing the apical aspect of the epithelium. Substitution of apical Na+ with NMDG+, a cation that fails to permeate most Na+- and/or K+-selective channels, alters the kinetic profile for NC-1059-induced effects (Fig. 6B) compared with a paired control (Fig. 6A). Exposure to NC-1059 was associated with a rapid increase in Isc that reached a maximum in <2 min (i.e., more rapidly than in control conditions) and then reversed polarity to achieve strong negative Isc (note that there is a break in the ordinate) for the duration of the experiment. Likewise, substitution of Cl by gluconate is associated with quantitative and qualitative differences in the NC-1059 response profile (Fig. 6C). NC-1059 causes a substantially larger increase in Isc than in control conditions, and the elevated Isc is maintained for the duration of the experiment. Finally, substitution of both Na+ and Cl provides a response profile that more closely approximates the control conditions (Fig. 6D). Regardless of the ions present in the apical solution, similar profiles for the increase in gTE are observed (Fig. 6E). Together, the results are consistent with NC-1059 being a nonselective or modestly anion-selective channel at the apical membrane that subsequently induces the operation of a pathway that allows for selective permeation by Na+ and Cl relative to NMDG+ and gluconate, respectively. These conclusions are based on the observations that, in the absence of apical Na+ and with no anion gradient, one might expect Na+ secretion through a nonselective ion channel. Such activity would result in a negative Isc. However, Isc initially increases in response to NC-1059, which is consistent with anion secretion but not cation secretion. Subsequently, Isc becomes negative, consistent with gradient-driven Na+ secretion through a pathway that is selective for Na+ over NMDG+. In this condition, any ongoing anion secretion would reduce the Isc magnitude. In the absence of apical Cl, the acute effect of NC-1059 on Isc is enhanced, as would be expected for an increased electrochemical driving force for anion secretion. Isc reaches a transient plateau and then continues to increase. A sustained elevation in Isc that is consistent with ongoing anion secretion (likely both gradient-driven and active transport) is observed throughout the experiment. Finally, in the absence of both Na+ and Cl in the apical solution (where similar gradient-driven forces for Na+ and Cl secretion would be present), the response profile is similar to the control conditions in which concentration gradients are not present. gTE does not increase to the same magnitude in Cl-free conditions, an outcome that would be expected if a portion of the conductance change depended on permeation through Cl-selective channels. Together the results suggest that the initial increase in Isc reflects anion (i.e., Cl) secretion, whereas extended effects exhibit little selectivity between Cl and Na+, although larger anions (e.g., gluconate) and cations (e.g., NMDG+) are less permeant through this pathway.



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Fig. 6. NC-1059-induced changes in Isc and gTE are affected by apical monovalent ion composition. Paired monolayers were exposed to NC-1059 (100 µM) as indicated in the presence or nominal absence of the indicated ions in the apical solution. Na+ and Cl were substituted by NMDG+ and gluconate, respectively, to maintain total electrolyte strength and osmolality (solution composition defined in materials and methods). Normal Ringer solution bathed the basolateral aspect of the epithelia throughout. AD: Isc in each of the indicated conditions. Note a break in the ordinate axis of B and C. E: gTE calculated for each voltage pulse in AD. 1-EBIO (300 µM) was present in all experiments. Dashed lines represent either zero current or zero conductance. Results are typical from 4 similar paired experiments.

 
NC-1059 is modestly Cl permselective. Xenopus oocytes have been employed to further test for permselectivity of NC-1059 ion channels in the absence of confounding effects associated with permeation through a noncellular (i.e., paracellular) pathway and the inability to set the electrochemical driving force. Typical results showing the NC-1059-induced increase in membrane conductance and ion-dependent change in Vrev are presented in Fig. 7, A and B, with results from numerous experiments being summarized in Fig. 7, C and D. The results indicate that exposure to NC-1059 is associated with a >18 ± 4-fold increase in membrane conductance (n = 6). Concomitant reduction in bath Na+ and Cl is associated with a rightward shift in Vrev of 5.2 ± 0.6 mV. Substitution of Cl by gluconate is associated with a greater rightward shift (8.4 ± 2.1 mV), whereas substitution of Na+ by NMDG+ is associated with a 4.2 ± 1.7-mV leftward shift. A synthetic peptide of similar amino acid composition, but in random order, has no effect on membrane conductance. Similarly, changes in bath ion composition have little effect on membrane conductance or on Vrev in the absence of NC-1059 (not shown). Together, these results indicate that the NC-1059-induced conductance has a finite permeability for both Cl and Na+. There is, however, little permselectivity between these monovalent ions. Mathematical analysis employing Eq. 1 indicates a Cl-to-Na+ permselectivity of 1.29 ± 0.04. Experiments in which these small monovalent ions are singly substituted support this conclusion in that a greater rightward shift in Vrev is observed with Cl substitution than the leftward shift observed with Na+ substitution. Oocyte results support observations made with intact monolayers by functionally demonstrating the membrane insertion of a permeation pathway that is modestly selective for Cl over Na+. Results from this assay do not, however, address the possibility that a paracellular pathway might be directly or indirectly affected by peptide exposure.



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Fig. 7. NC-1059 causes a Cl-selective conductance across membranes of Xenopus oocytes. A: typical overlays demonstrating currents recorded at nine voltages ranging from –100 to +60 mV in the absence or presence of NC-1059 (100 µM) in the presence of high (i.e., typical) NaCl and NC-1059-associated currents recorded in reduced (by ~90%) NaCl. B: current-voltage (I-V) relationships for the 3 conditions depicted in A. Solid lines represent the least-squares fit of linear function to the data sets from which slope conductance (g) and reversal potential (Vrev) are derived. Values are reported adjacent to each overlay. C: summary from 6 oocytes of derived slope conductances in the absence and presence of NC-1059 (100 µM) in typical Na+ and Cl concentrations. D: summary from 5 oocytes of changes in Vrev associated with concomitant reduction in Na+ and Cl, reduced Cl, and reduced Na+. Positive values indicate a rightward shift in Vrev. Changes in Vrev are consistent with the following selectivities: Cl > Na+, Cl > gluconate, and Na+ > NMDG+. Complete solution compositions and selectivity ratios are presented in the text.

 
NC-1059 allows permeation of 9.5-kDa FITC-dextran conjugate. Experiments have been conducted to determine if NC-1059-induced changes in gTE are mirrored by changes in permeation of larger, nonionic solutes. As shown in Fig. 8, NC-1059 exposure caused a substantial increment in transepithelial flux of FITC-labeled 9.5-kDa dextran over a 60-min assay period, although it was less than half of that observed across paired monolayers exposed to 3 mM EDTA in hypotonic Ringer solution (50% dilution with H2O). Tissue culture inserts are permeant to all sizes of dextran tested (up to 2.5 MDa; Fig. 8, inset). These results demonstrate that the NC-1059-stimulated increase in gTE is paralleled by an increase in concentration gradient-driven transepithelial flux of large, uncharged solutes. The lack of permeation by 77-kDa and larger solutes suggests that the NC-1059-associated permeation pathway has a finite maximal diameter or that the pathway exhibits some form of selectivity, an observation that is consistent with Isc measurements reported above for bi-ionic conditions.



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Fig. 8. Apical application of NC-1059 (100 µM) allows a 9.5-kDa FITC-dextran conjugate to permeate an MDCK monolayer while excluding larger molecular weight conjugates. Application of EDTA (3 mM) in hypotonic Ringer solution (50% dilution) allows access to the basolateral compartment for all FITC-dextran conjugates tested. Permeation across paired monolayers in the absence of treatment was subtracted to show treatment-induced changes in dye permeation. Assay was performed in triplicate, and results are typical from 3 similar experiments. Inset: permeation of FITC-conjugated dextran across a tissue culture support in the absence of cells. FITC-dextran conjugates permeate the tissue culture support at ~1,000-fold higher rate than in the presence of cells.

 
NC-1059-treated monolayers are transiently permeant to 9.5-kDa dextran. Experiments have been conducted to determine whether, like changes in gTE, the NC-1059-induced increase in permeability to 9.5-kDa dextran is both reversible and repeatable. Results presented in Fig. 9 verify earlier observations by showing that the two treatments, NC-1059 and EDTA/hypotonic Ringer solution, are associated with elevated permeation of 9.5-kDa FITC-dextran conjugate (compare results of initial treatment). All monolayers were washed after the response was assessed and were returned to the incubator in typical medium for subsequent experiments 2 days later. Monolayers from each of the three initial treatments were divided and exposed in parallel to each of the three treatments. Results demonstrate that, regardless of initial treatment and the magnitude of dye permeation, 2 days of recovery allow for the reformation of tight epithelial barrier. In each case, when monolayers were exposed to control conditions, a minimal amount of dextran permeation was observed, indicating that dextran permeability returns to a value that is indistinguishable from untreated monolayers within 2 days. Furthermore, the results demonstrate that the results of the three treatments are not affected by previous exposure to either NC-1059 or EDTA. In each treatment group, EDTA exposure was associated with the highest level of permeation. Permeation in the presence of NC-1059 is significantly greater than control but less than EDTA in each treatment group. The results show that apical NC-1059 exposure of MDCK monolayers causes a transient increase in permeability to uncharged solutes of up to 9.5 kDa, with no long-term deficit in epithelial barrier function observed.



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Fig. 9. NC-1059-induced FITC-conjugated dextran (9.5 kDa) permeation across MDCK epithelial cell monolayers is reversible and repeatable. Dextran permeation was evaluated across MDCK cell monolayers that were treated on day 1 with NC-1059 (100 µM), EDTA (6 mM in hypotonic Ringer solution), or an untreated control monolayer (12 monolayers per condition assayed in triplicate). Cell monolayers were washed and incubated for 2 days in culture media, and then subsets from each previous treatment (4 monolayers per condition) were exposed to 1 of the 3 treatments. Results are typical from 2 similar experiments.

 
Distribution of tight junction-associated proteins is unaltered by exposure to NC-1059. Data presented in Fig. 10 show the immunolocalization of tight junction-associated proteins using confocal microscopy. Protein components of the tight junction selected for analysis were actin, a cytoskeletal component that forms the junction-associated actomyosin ring; ZO-1, a putative scaffold protein that anchors the junctional complex to the cytoskeleton; and occludin, a transmembrane protein involved in tight junction formation. Compared with untreated controls, the distribution of actin, occludin, and ZO-1 immunoreactivity in MDCK monolayers was unchanged by exposure to NC-1059. Dense immunoreactivity for each tight junction constituent is observed to circumscribe all epithelial cells when viewed en face. Additionally, punctate intracellular occludin immunoreactivity is observed at the same focal plane, and diffuse ZO-1 immunoreactivity that appears to be associated with the nucleus is observed. Alternatively, exposure of epithelial cell monolayers to EDTA and hypotonic Ringer solution, a solution commonly employed to reduce junctional integrity and to disrupt cells from culture substrates, is associated with profound changes in localization of the perijunctional actin ring as well as the distribution of ZO-1. EDTA-treated cells appear to have "rounded up," and vacant areas in the field suggest that some cell sloughing occurs with this treatment.1ZO-1 immunoreactivity remains largely associated with the cell membrane but appears to be somewhat more diffuse than peptide-treated or untreated controls. Protein distribution in monolayers exposed to a non-channel-forming peptide (scrambled) of similar amino acid composition to NC-1059 is indistinguishable from that observed in vehicle-treated control monolayers, with cell-to-cell contacts apparently being maintained. These results suggest that the NC-1059-associated change in gTE results from tightly controlled changes in the paracellular pathway because large, uncharged solutes can readily permeate the epithelium without any apparent change in the distribution of tight junction-associated proteins or any indication of cell sloughing.



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Fig. 10. Indirect immunofluorescence of tight junction proteins [left, actin; center, occludin; right, zonula occludens-1 (ZO-1)] in MDCK cell monolayers following exposure to 1 of 4 treatments, as indicated: NC-1059 (100 µM), EDTA (6 mM in hypotonic Ringer solution), scrambled (peptide of similar amino acid composition to NC-1059 but in random order; 100 µM), untreated. Scale bar = 25 µm.

 

    DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
NC-1059 was synthesized as part of an ongoing program to develop synthetic peptides that form anion-selective channels in epithelial monolayers as potential therapeutics for CF. The rationale for the synthesis of this peptide was to determine if separate domains contribute to aggregation in aqueous solution and assembly of the peptide in cell membranes (3). This peptide is remarkable in that it remains monomeric in aqueous solution yet partitions into cell membranes and supports ion transport across the cell membrane and thus across epithelial cell monolayers. NC-1059 acts to increase the Isc across MDCK monolayers, with a K1/2 of ~40 µM, which is fourfold less than that of NK4-M2GlyR (2), a sequence from which it was developed. Observations made during these experiments initially suggested that NC-1059 also modulates epithelial tight junctions. Although not a target of the design process, this effect holds great therapeutic and research potential.

NC-1059 induces a concentration-dependent increase in Isc across epithelial cell monolayers with a concurrent increase in gTE. This peptide is the only sequence designed so far that has demonstrated the ability to increase gTE to this magnitude, which is in excess of conductance changes expected for apical channel formation, because related peptide sequences provide comparable increases in Isc but do not exhibit the dramatic effects on transmural conductance (3). This additional functionality suggests that the ability of the peptide to support anion secretion is separate from, but perhaps related to, the effect on gTE.

The mechanism by which NC-1059 modulates gTE is unclear. The simplest interpretation, that NC-1059 forms conductive pores in the apical membrane that fully account for the change in gTE, is inadequate. Similar peptide sequences (i.e., with the first 16 amino acid residues being identical) cause an equal increase in Isc across MDCK epithelial monolayers but do not affect transepithelial resistance to the same extent (3). Comparison of the data presented in Fig. 1D to an earlier report (3) shows that the concentration dependence for changes in gTE is right shifted compared with the concentration dependence of Isc. Additionally, permeation of 9.5-kDa dextran across the epithelium strongly suggests that a paracellular rather than a transcellular route is involved. A second simple possibility that can be discounted is that NC-1059 is cytotoxic and that a loss of cells accounts for the change in gTE (1). Visual inspection provides no indication that cells are absent from the epithelium following NC-1059 exposure as they are following EDTA exposure. Furthermore, tight junction proteins are not redistributed in response to NC-1059, and both the selectivity (Na+ > NMDG+; Cl > gluconate) and the finite size of the permeation pathway (9.5 but not 77 kDa dextran) suggest that a selective paracellular pathway is opened. Rather, the results suggest a specific interaction of NC-1059 with the cellular components involved in modulating gTE. This conclusion is bolstered by the "sidedness" of effects in that changes in electrical parameters are observed only with apical exposure. There is clearly precedence for metabotropic receptors selectively modulating the size exclusion of the paracellular pathway (21, 37), although evidence has not yet been acquired to suggest a metabotropic effect of NC-1059.

That NC-1059 is effective only from the apical aspect of the epithelium suggests that a "receptor-type" mechanism might be involved. There is at this time, however, no definitive evidence to support such a claim. Results presented in Fig. 2 might indicate that there is limited access of the peptide across the tissue culture support. However, the membrane was permeable to 2.5 MDa, and 77-kDa dextran permeated the membrane in the presence of cells following EGTA exposure. Thus size exclusion by the culture support is unlikely. An alternative to the "receptor" hypothesis is that the apical membrane exhibits a unique lipid milieu with which NC-1059 interacts. This possibility by necessity includes the supposition that a similar milieu must be present in Xenopus oocytes because NC-1059 quite effectively modulated membrane conductance in this system. A third possibility is that NC-1059 exhibits pleiotropic effects by interacting at multiple cellular sites. At this time it remains unclear whether channel formation (i.e., ion transport) is a prerequisite for modulation of gTE. The possibility exists that NC-1059 may interact with the apical membrane to form ion channels by mechanisms similar to those that have been indicated for closely related peptides and that effects on gTE require interaction with another epithelial target.

The NC-1059-induced change in gTE is transient in nature, reaching a peak value within the first 10–30 min of exposure. Several events might account for the transient nature of the response. It is possible that, due to charge neutralization or shielding, the peptide may undergo some aggregation and/or precipitation in Ringer solution (31), thus reducing the effective concentration. It was previously reported (3) that NC-1059 (initially termed NK4-Ala) does not aggregate in solution. However, the analysis was conducted with H2O as the solvent instead of Ringer solution. The ionic strength of the Ringer solution may promote peptide aggregation (a competing and irreversible reaction) that would tend to decrease the effective concentration of NC-1059 in the bath and in the cell membrane. Alternatively, the response may diminish due to protease degradation of the peptide, in turn due to uptake of peptide from the apical membrane that subsequently leads to proteolysis. Data presented in Fig. 4 suggest that peptide aggregation or inactivation cannot account for the transient nature of the effects. The possibility remains that there may be some downregulation of the metabolic process that modulates junction integrity. Data presented in Fig. 3 argue against this latter possibility in that the response to a submaximal concentration remains above baseline for at least 2 h and the epithelium subsequently responds to a higher concentration of NC-1059. Nonetheless, these and other possibilities may in part account for the transient nature of the response; additional experiments must be conducted to more fully evaluate these hypotheses.

There are numerous clinical situations in which modulation of an epithelial barrier presents therapeutic benefits. Drug absorption across intestinal, airway, or dermal epithelium could be enhanced with transient decreases in barrier function, making oral, inhaler, or topical administration of what are now parenteral drugs possible (9, 11). Oral or inhalant formulations of medications such as insulin would be less expensive to produce and more easily delivered than parenteral formulations (37). The permeability of the small intestinal epithelium to both insulin and immunoglobulin G was increased in rabbits when coadministered with ZOT (11). However, ZOT is limited in its therapeutic application because it is only effective in the small intestine (10). Additionally, ZOT is a 45-kDa protein that must be recombinantly produced and purified, although it has been shown that the majority of biological activity can reside in the COOH-terminal 15-amino acid segment (6). The M2GlyR-derived peptides are <3 kDa and can be prepared synthetically or recombinantly expressed.

Gene therapy for epithelium-associated diseases such as CF provides a second therapeutic setting in which modulation of transepithelial permeability is desirable (24, 32). Stable transfection of DNA sequences into epithelial cells in culture (7, 27) provides proof that genetic epithelial diseases can be treated or cured. However, bronchiolar epithelial cell viral receptors are located primarily in the basolateral membrane (17, 25, 38, 39), leading to a low efficiency of gene transfer from apical exposure to viral vectors. Thus high viral titers and long incubation times are required to increase transfection efficiency, which can lead to a decrease in the effectiveness of repeated treatments. Increased transfection efficiency has been achieved with some chemical modulators of tight junctions (e.g., EGTA, perfluorochemicals, fatty acids), although these treatments were sometimes associated with inflammation (5, 17). Modulators of epithelial barrier function would be the ideal agents to augment gene therapy, provided that they have a rapid onset, transient duration of action, and favorable safety profile, and do not decrease viral titer (35). Initial observations with NC-1059 suggest that it may fulfill these criteria, although additional experiments will be required to determine whether viral access is limited due to size exclusion (i.e., <77 kDa). Additional experiments would also be required to test for inflammation. In this regard it is encouraging that similar effects on gTE have been observed when an all-D-amino acid form of NC-1059 was used (unpublished observations).

NC-1059 also presents the potential for developing an increased understanding of physiological and pathophysiological processes that modulate tight junctions. Various epithelia throughout the body exhibit transepithelial electrical resistances that vary over four orders of magnitude (37), some of which change depending on the hormonal state (e.g., mammary; Refs. 22, 29) or physical environment (e.g., small intestine following a meal; Ref. 23). Signaling pathways that affect the paracellular pathway are not fully defined for any epithelium, and it is not known which mechanisms are broadly applicable and which are species or tissue specific. In this regard, it is noteworthy that NC-1059 stimulates gTE across male porcine reproductive epithelia, porcine ileal epithelia (IPEC-J2), and human colonic epithelia (Caco-2; unpublished observations). Thus, unlike ZOT, which affects only the small intestine (11), NC-1059 affects a broader spectrum of epithelia and, unlike Clostridium perfringens enterotoxin (19), causes no discernible cell damage. Thus NC-1059 can be used to survey a variety of tissues to identify common regulatory mechanisms.

In summary, NC-1059 is a channel-forming peptide that reversibly modulates conductance through the epithelial paracellular pathway. The response is fully repeatable and occurs without overt indications of cytotoxicity. This functionality provides a unique opportunity to conduct research regarding the mechanisms that selectively modulate tight junction maintenance. Additionally, NC-1059 represents a novel lead compound that might be developed to augment other forms of therapy that are currently limited by the epithelial barrier.


    GRANTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We gratefully acknowledge the assistance of Dr. Dan Boyle and the Kansas State University Biology Microscopy and Image Processing Facility, which has been supported in part by the Kansas National Science Foundation Experimental Program to Stimulate Competitive Research, by the Kansas National Aeronautics and Space Administration EPSCoR Program, by University resources, and by the Kansas Agricultural Experiment Station. This work is supported by National Institute of General Medicine Grants GM-43617 and GM-66620.


    ACKNOWLEDGMENTS
 
We thank Ryan Carlin, Suma Somasekharan, and Gary Radke for technical assistance.

This manuscript represents contribution number 03-126-J from the Kansas Agricultural Experiment Station.


    FOOTNOTES
 

Address for reprint requests and other correspondence: B. D. Schultz, Dept. of Anatomy and Physiology, 228 Coles Hall, Kansas State Univ., Manhattan, KS 66506 (E-mail: bschultz{at}vet.ksu.edu).

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.

1 Multiple peptide-treated and control monolayers were surveyed over numerous microscope fields to determine whether any evidence of cell sloughing could be identified in either of these conditions. A confluent monolayer was observed in all cases. Back


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
1. Bojarski C, Gitter AH, Bendfeldt K, Mankertz J, Schmitz H, Wagner S, Fromm M, and Schulzke JD. Permeability of human HT-29/B6 colonic epithelium as a function of apoptosis. J Physiol 535: 541–552, 2001.[Abstract/Free Full Text]

2. Broughman JR, Mitchell KE, Sedlacek RL, Iwamoto T, Tomich JM, and Schultz BD. NH2-terminal modification of a channel-forming peptide increases capacity for epithelial anion secretion. Am J Physiol Cell Physiol 280: C451–C458, 2001.[Abstract/Free Full Text]

3. Broughman JR, Shank LP, Takeguchi W, Schultz BD, Iwamoto T, Mitchell KE, and Tomich JM. Distinct structural elements that direct solution aggregation and membrane assembly in the channel-forming peptide M2GlyR. Biochemistry 41: 7350–7358, 2002.[CrossRef][ISI][Medline]

4. Citi S. Introduction: opening up tight junctions. Semin Cell Dev Biol 11: 277–279, 2000.[CrossRef][ISI][Medline]

5. Coyne CB, Kelly MM, Boucher RC, and Johnson LG. Enhanced epithelial gene transfer by modulation of tight junctions with sodium caprate. Am J Respir Cell Mol Biol 23: 602–609, 2000.[Abstract/Free Full Text]

6. Di Pierro M, Lu R, Uzzau S, Wang W, Margaretten K, Pazzani C, Maimone F, and Fasano A. Zonula occludens toxin structure-function analysis. Identification of the fragment biologically active on tight junctions and of the zonulin receptor binding domain. J Biol Chem 276: 19160–19165, 2001.[Abstract/Free Full Text]

7. Drumm ML, Pope HA, Cliff WH, Rommens JM, Marvin SA, Tsui LC, Collins FS, Frizzell RA, and Wilson JM. Correction of the cystic fibrosis defect in vitro by retrovirus-mediated gene transfer. Cell 62: 1227–1233, 1990.[ISI][Medline]

8. Edens HA and Parkos CA. Modulation of epithelial and endothelial paracellular permeability by leukocytes. Adv Drug Delivery Res 41: 315–328, 2000.[CrossRef][ISI][Medline]

9. Fasano A. Modulation of intestinal permeability: an innovative method of oral drug delivery for the treatment of inherited and acquired human diseases. Mol Genet Metab 64: 12–18, 1998.[CrossRef][ISI][Medline]

10. Fasano A. Novel approaches for oral delivery of macromolecules. J Pharm Sci 87: 1351–1356, 1998.[CrossRef][ISI][Medline]

11. Fasano A and Uzzau S. Modulation of intestinal tight junctions by Zonula occludens toxin permits enteral administration of insulin and other macromolecules in an animal model. J Clin Invest 99: 1158–1164, 1997.[Abstract/Free Full Text]

12. Gassler N, Rohr C, Schneider A, Kartenbeck J, Bach A, Obermuller N, Otto HF, and Autschbach F. Inflammatory bowel disease is associated with changes of enterocytic junctions. Am J Physiol Gastrointest Liver Physiol 281: G216–G228, 2001.[Abstract/Free Full Text]

13. Hille B. Ion channels of excitable membranes. Sunderland, MA: Sinauer, 1992.

14. Hopkins AM, Li D, Mrsny RJ, Walsh SV, and Nusrat A. Modulation of tight junction function by G protein-coupled events. Adv Drug Delivery Res 41: 329–340, 2000.[CrossRef][ISI][Medline]

15. Howard M, DuVall MD, Devor DC, Dong JY, Henze K, and Frizzell RA. Epitope tagging permits cell surface detection of functional CFTR. Am J Physiol Cell Physiol 269: C1565–C1576, 1995.[Abstract/Free Full Text]

16. Kleeff J, Shi X, Bode HP, Hoover K, Shrikhande S, Bryant PJ, Korc M, Buchler MW, and Friess H. Altered expression and localization of the tight junction protein ZO-1 in primary and metastatic pancreatic cancer. Pancreas 23: 259–265, 2001.[CrossRef][ISI][Medline]

17. Koehler DR, Hitt MM, and Hu J. Challenges and strategies for cystic fibrosis lung gene therapy. Mol Ther 4: 84–91, 2001.[CrossRef][ISI][Medline]

18. Li JC, Mruk D, and Cheng CY. The inter-Sertoli tight junction permeability barrier is regulated by the interplay of protein phosphatases and kinases: an in vitro study. J Androl 22: 847–856, 2001.[Abstract/Free Full Text]

19. McClane BA. The complex interactions between Clostridium perfringens enterotoxin and epithelial tight junctions. Toxicon 39: 1781–1791, 2001.[CrossRef][ISI][Medline]

20. Mitomi H, Tanabe S, Igarashi M, Katsumata T, Arai N, Kikuchi S, Kiyohashi A, and Okayasu I. Autoimmune enteropathy with severe atrophic gastritis and colitis in an adult: proposal of a generalized autoimmune disorder of the alimentary tract. Scand J Gastroenterol 33: 716–720, 1998.[CrossRef][ISI][Medline]

21. Mullin JM, Marano CW, Laughlin KV, Nuciglio M, Stevenson BR, and Soler P. Different size limitations for increased transepithelial paracellular solute flux across phorbol ester and tumor necrosis factor-treated epithelial cell sheets. J Cell Physiol 171: 226–233, 1997.[CrossRef][ISI][Medline]

22. Nguyen DA and Neville MC. Tight junction regulation in the mammary gland. J Mammary Gland Biol Neoplasia 3: 233–246, 1998.[CrossRef][ISI][Medline]

23. Nusrat A, Turner JR, and Madara JL. Molecular physiology and pathophysiology of tight junctions. IV. Regulation of tight junctions by extracellular stimuli: nutrients, cytokines, and immune cells. Am J Physiol Gastrointest Liver Physiol 279: G851–G857, 2000.[Abstract/Free Full Text]

24. O'Neal WK and Beaudet AL. Somatic gene therapy for cystic fibrosis. Hum Mol Genet 3: 1497–1502, 1994.[Abstract]

25. Pickles RJ, McCarty D, Matsui H, Hart PJ, Randell SH, and Boucher RC. Limited entry of adenovirus vectors into well-differentiated airway epithelium is responsible for inefficient gene transfer. J Virol 72: 6014–6023, 1998.[Abstract/Free Full Text]

26. Reddy GL, Iwamoto T, Tomich JM, and Montal M. Synthetic peptides and four-helix bundle proteins as model systems for the pore-forming structure of channel proteins. II. Transmembrane segment M2 of the brain glycine receptor is a plausible candidate for the pore-lining structure. J Biol Chem 268: 14608–14615, 1993.[Abstract/Free Full Text]

27. Rich DP, Anderson MP, Gregory RJ, Cheng SH, Paul S, Jefferson DM, McCann JD, Klinger KW, Smith AE, and Welsh MJ. Expression of cystic fibrosis transmembrane conductance regulator corrects defective chloride channel regulation in cystic fibrosis airway epithelial cells. Nature 347: 358–363, 1990.[CrossRef][ISI][Medline]

28. Schultz BD, Takahashi A, Liu C, Frizzell RA, and Howard M. FLAG epitope positioned in an external loop preserves normal biophysical properties of CFTR. Am J Physiol Cell Physiol 273: C2080–C2089, 1997.[Abstract/Free Full Text]

29. Stelwagen K, McFadden HA, and Demmer J. Prolactin, alone or in combination with glucocorticoids, enhances tight junction formation and expression of the tight junction protein occludin in mammary cells. Mol Cell Endocrinol 156: 55–61, 1999.[CrossRef][ISI][Medline]

30. Takahashi A, Watkins SC, Howard M, and Frizzell RA. CFTR-dependent membrane insertion is linked to stimulation of the CFTR chloride conductance. Am J Physiol Cell Physiol 271: C1887–C1894, 1996.[Abstract/Free Full Text]

31. Tomich JM, Wallace D, Henderson K, Mitchell KE, Radke G, Brandt R, Ambler CA, Scott AJ, Grantham J, Sullivan L, and Iwamoto T. Aqueous solubilization of transmembrane peptide sequences with retention of membrane insertion and function. Biophys J 74: 256–267, 1998.[Abstract/Free Full Text]

32. Wagner JA and Gardner P. Toward cystic fibrosis gene therapy. Annu Rev Med 48: 203–216, 1997.[CrossRef][ISI][Medline]

33. Wallace DP, Tomich JM, Iwamoto T, Henderson K, Grantham JJ, and Sullivan LP. A synthetic peptide derived from glycine-gated Cl channel induces transepithelial Cl and fluid secretion. Am J Physiol Cell Physiol 272: C1672–C1679, 1997.[Abstract/Free Full Text]

34. Walsh SV, Hopkins AM, and Nusrat A. Modulation of tight junction structure and function by cytokines. Adv Drug Delivery Res 41: 303–313, 2000.[CrossRef][ISI][Medline]

35. Wang G, Zabner J, Deering C, Launspach J, Shao J, Bodner M, Jolly DJ, Davidson BL, and McCray PB Jr. Increasing epithelial junction permeability enhances gene transfer to airway epithelia In vivo. Am J Respir Cell Mol Biol 22: 129–138, 2000.[Abstract/Free Full Text]

36. Wang W, Uzzau S, Goldblum SE, and Fasano A. Human zonulin, a potential modulator of intestinal tight junctions. J Cell Sci 113: 4435–4440, 2000.[Abstract/Free Full Text]

37. Ward PD, Tippin TK, and Thakker DR. Enhancing paracellular permeability by modulating epithelial tight junctions. Pharm Sci Technol Today 3: 346–358, 2000.[CrossRef][Medline]

38. Zabner J, Freimuth P, Puga A, Fabrega A, and Welsh MJ. Lack of high affinity fiber receptor activity explains the resistance of ciliated airway epithelia to adenovirus infection. J Clin Invest 100: 1144–1149, 1997.[Abstract/Free Full Text]

39. Zabner J, Zeiher BG, Friedman E, and Welsh MJ. Adenovirus-mediated gene transfer to ciliated airway epithelia requires prolonged incubation time. J Virol 70: 6994–7003, 1996.[Abstract]





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