Two-path impedance spectroscopy for measuring paracellular and transcellular epithelial resistance

Abstract

Solutes are transported across epithelial cell layers via transcellular and paracellular pathways. The transcellular pathway leads across the apical and basolateral cell membrane, whereas the paracellular pathway is directed through the tight junction. Tight junction proteins (claudins, occludin, tricellulin) can not only form barriers but also paracellular channels that are--in concert with membrane channels and transporters--regulated in a wide range in health and disease states. Thus, it is desirable to determine para- and transcellular resistance (R(para), R(trans)) separately. This cannot be achieved by conventional transepithelial resistance (TER) measurements. We present an impedance spectroscopic approach that is optimized for differentiation between these two pathways. The method is based on a transepithelial impedance measurement in specialized Ussing chambers, combined with a Ca(2+)-dependent modulation of R(para) through EGTA and flux measurements of a nonradioactive paracellular marker, fluorescein. The prerequisites are a paracellular marker that varies in parallel to 1/R(para), an experimental regime that alters R(para) without affecting R(trans), and exact knowledge of the resistance of subepithelial components. The underlying prerequisites and the applicability as a routine method were verified on cell lines of different tightness including HT-29/B6 colon cells and Madin-Darby canine kidney tubule cells C7 and C11.

Figure 1

(A) Equivalent electrical circuit for one path impedance measurements, that only discriminate between subepithelial (R^sub) and epithelial (R^epi) resistance, and the epithelial capacity C^epi. (B) Equivalent electrical circuit for two path impedance measurements used in this study. Here, the epithelial resistance R^epi is composed of a paracellular (R^para) and a parallel transcellular (R^trans) resistance. Under DC conditions or AC conditions at very low frequencies R^epi = R^para × R^trans/(R^para + R^trans). (C) Equivalent electrical circuit for a model discriminating between the apical (index ap) and the basolateral (index bl) side of an epithelium. In this model, the capacitance C^epi of the models presented in A and B is composed of C^ap and C^bl (C^epi = C^ap × C^bl/(C^ap + C^bl). Under DC conditions or AC conditions at very low frequencies R^epi = R^para × (R^ap + R^bl)/(R^para + R^ap + R^bl). (D) Nyquist diagram (plot of the real and the imaginary portion of the impedance, Z_re, Z_im) for models shown in A and B. For impedance values measured at different AC frequencies, both models result in a semicircle. For high frequencies (ω → ∞) Z_re approaches R^sub, for low frequencies (ω → 0) Z_re approaches R^t = R^sub + R^epi. The capacitance C^epi can be calculated from the frequency at which |Z_im| reaches a maximum (C^epi = 1/(ω_|Zim|max × R^epi). Nyquist diagrams for the model depicted in C only yield a semicircle, if C^ap/C^bl = R^bl/R^ap (i.e., if the membrane time constant τ = R × C is constant).

Figure 2

(A) Plotting G^epi versus the flux of a paracellular marker under experimental conditions that only modify the paracellular pathway yields a straight line with the slope s and the y-intercept G^trans. G^para is calculated as s × Flux. (B) Varying G^trans shifts the straight line along the y axis, whereas changing G^para moves the values along the straight line (open circles). A transcellular flux component of the marker shifts the straight line along the x axis. (C) Changes in the general permeability properties of the paracellular pathway affect the slope of the straight line. (D) Over- or underestimation of R^sub distorts the relationship between flux and G^epi.

Figure 3

Nyquist plot of impedance spectra recorded from a HT-29/B6 cell layer in the absence and presence of EGTA (A and C) and forskolin (B and D). (A) Parameters (mean ± SE) obtained from the three control spectra are R^sub, 5.80 ± 0.13 Ω × cm²; R^epi, 615.5 ± 9.4 Ω × cm²; C^epi, 4.63 ± 0.03 μF/cm². Simultaneously measured fluorescein flux increased from 0.10 ± 0.01 nmol/cm²/h (n = 3) under control conditions by a factor of 20 at an R^epi_−EGTA of 56 Ω × cm². This allowed to calculate R^trans to 1300 Ω × cm², $R_{\mathrm{control}}^{\text{para}}$ to 1170 Ω × cm² and $R_{\mathrm{EGTA}}^{\text{para}}$ to 58.5 Ω × cm² (Eqs. 1 and 2). (B) Parameters (mean ± SE) obtained from the three control spectra: R^sub, 6.90 ± 0.11 Ω × cm²; R^epi, 618.5 ± 12.8 Ω × cm²; C^epi, 3.93 ± 0.04 μF/cm². The simultaneously measured fluorescein flux remained virtually unchanged (control, 0.31 ± 0.05 nmol/cm²/h; forskolin 0.26 ± 0.03 nmol/cm²/h). Without further information, R^trans and R^para cannot be determined from these data. (C and D) Same as A and B, but normalized with respect to R^epi to illustrate, that changes in the paracellular pathway (EGTA application, C) did not alter the general shape of the curves. In contrast, changing the transcellular pathway by forskolin-induced Cl⁻ secretion, greatly alters the general shape of the curves (D).

Figure 4

G^epi versus fluorescein flux plots from experiments on HT-29/B6 cell layers in the absence and presence of EGTA (A) and forskolin followed by EGTA (B). (A) Values obtained in the absence (•) and presence (○) of EGTA fall onto a straight line. G^trans estimated from the y-intercept: 0.89 mS/cm² (R^trans = 1120 Ω × cm²). Line, linear regression; slope 10.7 × 10⁶ S × h/mol. (B) Values obtained after the application of forskolin (■) are shifted along the y axis compared to values obtained under control conditions (□), whereas values in the presence of forskolin and forskolin + EGTA (▴) fall onto a straight line (solid line: linear regression, slope 9.1 × 10⁶ S × h/mol). This line has a similar slope as the line obtained in the absence of forskolin obtained in A (dotted line). Thus, in the presence of forskolin, G^trans increased to 3.0 mS/cm² (R^trans = 333 Ω × cm²).

Figure 5

(A) G^epi versus fluorescein flux was independent of temperature in the range of 15°C to 37°C. Line, linear regression; slopes 7.4 × 10⁶ S × h/mol (37°C), 7.0 × 10⁶ S × h/mol (25°C). (B) G^epi versus fluorescein flux was independent of the flux direction (basolateral to apical, ♦; apical to basolateral, ♢, slopes of regression lines 7.5 × 10⁶ S × h/mol and 7.4 × 10⁶ S × h/mol, respectively). (C) In contrast, G^epi versus Na⁺ flux was clearly dependent of the flux direction (basolateral to apical, •; apical to basolateral, ○). Slopes of regression lines were similar (2.03 × 10³ S × h/mol and 2.01 × 10³ S × h/mol, respectively) but values in the apical to basolateral direction were shifted along the x axis, indicating a transcellular Na⁺ flux under these conditions. G^trans calculated from data in the basolateral to apical direction was similar to G^trans calculated from fluorescein flux (Fig. 4A) and amounted to 1.05 mS/cm² (R^trans = 950 Ω × cm²), whereas in the apical to basolateral direction a calculation of G^trans would yield a value of −2.42 mS/cm² (R^trans = −410 Ω × cm²). (D) Comparison of G^trans versus flux of fluorescein (♢), Na⁺ (basolateral to apical, ♦) and Cl⁻ (apical to basolateral, ▴). The y-intercepts under these three conditions result in a mean G^trans of 0.88 mS/cm² (R^trans = 1140 Ω × cm²). The slopes for Na⁺ (1.029 × 10³ S × s/cm³) and Cl⁻ flux (1.031 × 10³ S × s/cm³) were very similar, indicating, that HT-29/B6 cell layers do not discriminate between these two ions. The slope for fluorescein was larger (3.9 × 10³ S × s/cm³), i.e., fluorescein permeability lower, as expected for an ion of larger diameter. (E) In contrast to fluorescein, G^epi versus 10 kDa FITC-dextran flux did not result in a straight line, indicating that 10 kDa FITC-dextran does not pass through the same paracellular pores as the ions determining paracellular conductance. (F) In Caco-2 cell layers G^epi versus fluorescein flux (apical to basolateral) did not yield a straight line (values the absence ■ and presence □ of EGTA). Therefore, fluorescein flux should not be used to estimate G^trans in these cells.

Figure 6

(A) G^epi versus fluorescein permeability plots from experiments on MDCK C11 cell layers. Values obtained in the absence (♢) and presence (♦) of EGTA fall onto a straight line. G^trans estimated from the y-intercept: 15.4 mS/cm² (R^trans = 64.9 Ω × cm²). Line: linear regression, slope 2.12 × 10³ S × s/cm³. (B) G^epi versus fluorescein permeability plots from experiments on MDCK C7 cell layers. Values obtained in the absence of EGTA (□) do not fall onto the same line as those obtained in the presence of EGTA (■). Estimates from the y-intercept of the linear regression (solid line, slope 2.05 × 10³ S × s/cm³) of the values obtained in the presence of EGTA yielded a G^trans of 1.44 mS/cm², but values in the absence of EGTA lie ∼1 mS/cm² lower, as judged by shifting the regression line along the y axis (dotted line). (C) G^epi versus Cl⁻ permeability plots from experiments on MDCK C7 cell layers. As in B, values obtained in the absence of EGTA (▵) lie ∼1 mS/cm² lower than estimated from a linear regression (solid line, slope 0.66 × 10³ S × s/cm³) of values obtained in the presence of EGTA (▴). In the presence of EGTA a G^trans of 1.41 mS/cm² is calculated. (D) G^epi versus Na⁺ permeability plots from experiments on MDCK C7 cell layers. As in B and C, values obtained in the absence of EGTA (♢) lie ∼1 mS/cm² lower than estimated from a linear regression (solid line, slope 0.63 × 10³ S × s/cm³) of values obtained in the presence of EGTA (♦). In the presence of EGTA a G^trans of 1.1 mS/cm² is calculated. Thus, from B-D, the mean G^trans in the absence of EGTA amounts to 0.31 mS/cm², yielding a R^trans of 3230 Ω × cm². (E) In MDCK C7 cells stably transfected with the paracellular cation pore claudin-10b, a plot of G^epi versus fluorescein (▴), Cl⁻ (○), and Na⁺ (♦) permeability yields G^trans values of 0.94, 0.93, and 0.98 mS/cm², respectively, which have to be corrected by 0.6 mS/cm² for values in the absence of EGTA. R^trans thus amounts to 2860 Ω × cm². The slope of G^epi versus Na⁺ (0.50 × 10³ S × s/cm³) is lower than for Cl⁻ (1.12 × 10³ S × s/cm³) and fluorescein (3.3 × 10³ S × s/cm³), mirroring the claudin-10b-induced increased in cation. (F) An atypical clone of MDCK II cells stably transfected with claudin-3 clone presented a strongly reduced G^trans in the absence of EGTA (♦) whereas in the presence of EGTA (▴), G^trans (13.4 mS/cm²) was comparable to the G^trans obtained in control cells shown in A. Solid line: linear regression (slope 5.9 × 10⁶ S × h/mol); dotted line: regression line shifted along the y axis.

Figure 7

(A) G^epi versus fluorescein permeability plot from experiments on HT-29/B6 cell layers in the absence and presence of EGTA. G^trans and G^para from different experiments (points joined by lines for clarity; see Fig. S3 for regression lines) can be evaluated separately to obtain mean resistances, if the general plot of all values shows no effects of EGTA on G^trans. If, as in MDCK C7 cells, EGTA has an effect on G^trans, values have to be corrected (Fig. 6). (B) Mean ± SE of R^para, R^trans, and R^epi calculated from single experiments (HT-29/B6, n = 8; MDCK C7, n = 6; MDCK C11, n = 5) as described in A. A ratio R^para/R^trans >1 indicates a (moderately) tight epithelium (HT-29/B6, MDCK C7), a ratio ≤1 a leaky epithelium (MDCK C11). (C) Mean ± SE of the epithelial capacities in HT-29/B6 and MDCK cell layers. Capacity values per unit filter area are significantly higher in HT-29/B6 than in both MDCK C7 and C11 cells (p < 0.01, Student's t-test), indicating a stronger increase in surface area in these cells, e.g., through microvilli or membrane invaginations. R^sub was similar for all cell types (HT-29/B6, 10.9 ± 1.4 Ω × cm²; MDCK C7, 11.4 ± 1.2 Ω × cm²; MDCK C11, 10.7 ± 1.6 Ω × cm²) and is assumed to primarily reflect the resistance of the filter supports.