Frequency spectrum of transepithelial potential difference reveals transport-related oscillations

Abstract

How epithelia transport fluid is a fundamental issue that is unresolved. Explanations offered include molecular engines, local transcellular osmosis, local paracellular osmosis, and paracellular fluid transport. On the basis of experimental and theoretical work done on corneal endothelium, a fluid transporting epithelium, we suggest electroosmotic coupling at the level of the intercellular junctions driven by the transendothelial electrical potential difference as an explanation of paracellular fluid transport. We collect frequency spectra of that potential difference in real-time. For what we believe is the first time for any epithelium, we report that, unexpectedly, the potential difference displays oscillations at many characteristic frequencies. We also show that on both stimulating cell activity and inhibiting ion transport mechanisms, there are corresponding changes in the oscillations amplitudes that mirror changes known previously in rates of fluid transport. We believe these findings provide a novel tool to study the kinetics of electrogenic elements such as channels and transporters, which from this evidence would give rise to current oscillations with characteristic periods going from 150 ms to 8 s.

Figure 1

Schematic diagram of the Ussing chamber and instruments used. Cis, endothelial side; trans, stromal side. Temperature-control jackets are not shown. They surround the bottom chambers (Lucite) and the aeration funnels (glass). A stainless-steel hemispherical wire net (not shown) on the trans hemichamber supports the corneal stroma.

Figure 2

Transendothelial electrical potential difference as a function of time. The upper curve (TEPD) shows a 10-min segment of a representative experiment (n = 43). The lower curve (CTRL) depicts a record of the potential difference obtained with the solution-filled chamber (R ∼ 15 KΩ; n = 8).

Figure 3

Frequency spectrum of the TEPD. For the curve shown in the insert, the frequency spectra of 10 experiments were averaged (average multiple curves routine; Origin). In the main plot, only the frequency domain of most interest is shown. The upper curve (endothelium) shows an individual frequency spectrum of the TEPD. Numbers near the peaks denote their periods τ in s. The lower line (chamber) shows a control spectrum of the potential difference obtained with the solution-filled chamber (n = 87).

Figure 4

Effects of inhibition with ouabain. Curves are frequency spectra of TEPD before (solid line, control) and ∼10 min after the addition of 100 μM ouabain (dashed line, shaded area). Spectra are averaged (n = 6). Periods are shown for the corresponding highest low-frequency peaks (peak α); deviations are also shown for those peaks.

Figure 5

Effects of inhibition with DIDS. Frequency spectra of TEPD before (solid line, control) and ∼10 min after the addition of 100 μM DIDS (dashed line, shaded area). Curves represent the averages of eight experiments. Periods and deviations are shown for the α-peaks.

Figure 6

Effect of stimulation with ATP; averages of six experiments. Curves represent spectra under control conditions and 10 min after the addition of 100 μM ATP. Deviations are shown for the α-peaks. The area between the two curves (representing the stimulation by ATP) is shaded. An auxiliary horizontal line (dashed) is drawn at y = 3.6.

Figure 7

Bars denote averages of the periods for the α-peaks in the different groups of experiments. The vertical lines correspond to the average ± SE for all groups combined (5.3 ± 2.0 s).

Figure 8

Corneal endothelial plasma membrane transporters and channels. Locations have been previously discussed (9). This simplified scheme shows only elements relevant to transcellular ionic flows. Abbreviations: ch, channel; N3B, 3:1 HCO₃⁻-Na⁺ cotransporter; N2B, 2:1 HCO₃⁻-Na⁺ cotransporter; N, Na⁺; K, K⁺; B, HCO₃⁻; C, Cl⁻; H, H⁺. Electrogenic elements are in large lettering. The local (open circuit) recirculating current is also shown, with its direction marked by arrows. Na⁺ ions preferentially carry the current through the junctions.

Figure 9

Simulation of the effects of ouabain and DIDS on parameters of the corneal endothelium using our computer program described previously that models the endothelium (9). The runs were set so that after a 50-s control interval, the relative permeabilities of the target elements changed as follows: for the ouabain case, the Na⁺/K⁺ ATPase from 1 to 0.0001; for the DIDS case, (a) the apical Na⁺-HCO₃⁻ cotransporter (N3B), (b) the basolateral Na⁺-HCO₃⁻ cotransporter (N2B), and (c) the basolateral Cl⁻/HCO₃⁻ exchanger, all from 1 to 0.01. For the ATP case, the relative permeabilities were increased by 50% for: (a) the Na⁺/K⁺ ATPase; (b) the N3B; (c) the N2B; (d) the Na⁺ channel, and (e) the Cl⁻ channel. The panels show the temporal changes in the relative turnover rates of (or fluxes through) several electrogenic elements of relevance. Na⁺ ch, Cl⁻ ch: Na⁺ and Cl⁻ channels. The simulations were run for 10 min after inhibition to approximate the times at which spectra were collected after exposure to the agents in actual experiments.