Ceil dissociation and culture

Adult newts (Cynops pyrrhogaster, 6-10 cm in body length) were anesthetized with 0.1% FA100 (4-allyl-2-methoexyphenol: Tanabe) and decapitated. Eyes were enucleated and sterilized with 70% ethanol. They were hemisected posterior to the ora serrata and the neural retinas together with lens were removed from the eye cups with fine forceps in control saline solution (in mM: NaCl 110; KCl 3.7; MgCI₂ 1.0; CaCI₂ 3.0; Na-pyruvate 1.0; glucose 13; HEPES 5). The eye cups were transferred to Ca2⁺- and Mg2⁺-free solution with 10 mM EGTA at pH 7.5 and then incubated at 25°C for about 20 min. The retinal pigment epithelium (RPE) was peeled off from the choroidal layers. Isolated RPEs were collected from several eyes and cut to pieces. Pieces of RPEs were incubated in control solution with 0.1% trypsin for 50 min at 28°C. After rinsing several times with 80% Leibovitz's L-15 medium (Flow Laboratories) to dilute the enzyme, RPE cells were dissociated by gentle mechanical trituration. About 500 μl of cell suspension medium was plated in 35 mm sterile plastic dishes (Falcon, No. 3001). The dish was previously coated with 1% concanavalin A (conA) for 2 hr, washed 5 times with distilled water and air-dried. Freshly dissociated RPE cells were first allowed to adhere for about 20 min, then rinsed a few times. Finally, about 2.5 ml of culture medium with 5% fetal calf serum (HyClone) was added to each dish. A density of cells attached on the substrate was 500∼800 cells/cm². All cultures were maintained at 25°C. The culture medium was not changed until cells were examined electro-physiologically.

Solutions and drugs

The dish containing dissociated RPE cells was mounted on the stage of an inverted microscope with phase-contrast optics (Olympus IMT-2). For physiological experiments, the culture medium was replaced with control saline solution described above. in Na⁺-free solution, NaCI was substituted with choline chloride. Test solutions containing tetrodotoxin (TTX), saxitoxin (STX) and tetraetylammonium (TEA) were made by adding an appropriate amount of these drugs directly to the control solution. The solution was fed into the dish by gravity at a rate of about 0.8 ml/min, and withdrawn by suction.

To arrest the cell cycle of RPE cells, we used trichostatin A (TSA) which was originally reported as fungistatic antibiotics (Tsuji et al., 1976) and recently demonstrated as a reversible inhibitor of the proliferation of mammalian cultured cells (Yoshida and Beppu,1988). This drug at concentrations of 0.1 ∼1.0 μg/ml was added in the solution at the beginning of cell culture.

Electrical measurements

Electrophysiological recordings were made from freshiy dissociated or cultured RPE cells using whole-cell patch-clamp techniques as described (Hamill et al., 1981 ). The tip diameter of the recording glass pipette was 1-2 μm, and the resistance was 2-5 MΩ in the bath solution. The recording pipettes were filled with the following solution (mM): KCl 138, HEPES 10, EGTA 5. The pipette was connected to the patch-clamp amplifier (Nihon Kohden, CEZ-2300). The bath was grounded with an Ag-AgCI wire. Junction potentials at the pipette tip were nulled by an offset current sufficient to make the output current zero. The membrane current was filtered at 2 kHz (a 4-pole Bessel filter). The capacitative current transients were not corrected. Data were sampled and digitized with a 12-bit analog-to-digital converter connected to a microcomputer (NEC, PC-9801VX) and stored on floppy disks for later analysis.

Culture of pigment epithelial cells

Figure 1 illustrates a cultured RPE cell dissociated from adult newt retina under phase contrast microscopy. Freshly dissociated RPE cells are often gourd-shaped, and characterized by the presence of dense pigmentation in the apical portion of the cell and nonpigmented smooth membrane of the basal portion (Fig. 1A). The mean diameter of freshly dissociated RPE cells (long axis) was 39±5 μm (SD, n=31). The cells allowed to attach onto a substrate began to flatten within 1-2 days (Fig. 1B). In the following few days, they continued to flatten and then their nuclei (arrow) became visible, because pigment granules spreaded throughout the cytoplasm (Fig. 1C). The shape of stretched cells varied widely from cell to cell. It was spherical or ova! in some cases and slender in others. The first mitosis was observed after 6 days in culture (Fig. 1D).

Fig. 1.

Phase-contrast photomicrograph of a cultured newt retinal pigment epithelial (RPE) cell. A: RPE cell attached to the plastic dish 5 hours after plating. B: Cell after 2 days in culture. C: Cell after 5 days in culture. D: Cell after 6 days in culture. Arrow head shows a nucleus.

Voltage-gated K⁺ currents in freshly dissociated RPE cells

Figure 2 shows voltage and current records from a freshly dissociated RPE cell. The cell was initially hyperpolarized by a constant current injection to maintain the membrane potential around −80 mV. Depolarizing currents under current clamp conditions produced responses with an exponential voltage rise (passive responses) which were followed by a voltage-dependent decline in amplitude (Fig. 2A). Evidence that this voltage decline is due to activation of voltage-gated outward current is shown in Figure 2B and 2C where the same cell was voltage-clamped. When the cell was depolarized by a series of voltage pulses from −160 to +40 mV in 20 mV steps, the outward current was activated around −20 mV and exhibited a voltage-dependent increase (Fig. 2B). The same voltage protocol was repeated during superfusion with a medium containing 50 mM TEA (Fig. 2C). TEA suppressed the outward current. Since TEA is known as a blocker of the delayed outward K⁺ current in a variety of excitable cells (Hille, 1984), the result suggests that a major ion channel of freshly dissociated RPE cells is delayed K⁺ channel.

Fig. 2.

Voltage and current responses recorded from a freshly dissociated RPE cell. A: Voltage responses under current clamp conditions. The cell was initially hyperpolarized from a resting potential of −45 mV to −80 mV by a constant current injection of about 8 nA and then depolarizing current pulses were applied for 150 msec. Four sweeps were superimposed. A small current pulse produced a response with an exponential time course (passive response). Larger current pulses produced larger passive responses, followed by voltage-dependent decline in amplitude. B: Membrane currents recorded from the cell in control solution under voltage-clamp conditions. Holding potential was −80 mV. The current was evoked by a series of voltage pulses (150 msec) from −160 to +40 mV in 20 mV steps. C: Suppression of the voltage-gated outward currents by 50 mM TEA.

Expression of voltage-gated Na⁺ currents in cultured RPE cells

Within several days in culture, more than 50% of RPE cells examined exhibited immature action potentials and transient inward currents that were never observed in freshly dissociated cells. Figure 3 shows voltage and current records from a cell maintained in culture for 6 days. When depolarizing currents were applied under current clamp conditions, the slow-rising action potentials of long duration was initiated above −40 mV (Fig. 3A). The onset of the response was indicated by an arrow. The same cell was depolarized under voltage clamp conditions from a holding potential of −80 mV to +40 mV in 20 mV steps. As the membrane potential was depolarized above −40 mV, a transient inward current was activated (Fig. 3B). This current was suppressed by replacement of NaCI in the external solution with choline-CI (Fig. 3C). The result suggests that the inward current may be carried by Na⁺.

Fig. 3.

Voltage and current responses recorded from a RPE cell following 6 days in culture. A: Voltage responses under current clamp conditions. The cell was initially hyperpolarized from a resting potential of −56 mV to −80 mV by a constant current injection and then depolarizing current pulses were applied for 150 msec. Five sweeps were superimposed. Slow-rising action potential of long duration was initiated above −40 mV. An arrow points to the onset of the action potential. B: Membrane currents recorded from the cell in control solution under voltage-clamp conditions. Holding potential was −80 mV. The current was evoked by a series of voltage pulses (150 msec) from −80 mV to +40 mV in 20 mV steps. Voltage-gated transient inward current was activated above −40 mV. C: Suppression of the inward and outward currents in Na⁺-free solution containing 20 mM TEA.

TTX-insensitive Na⁺ current

Figure 4 illustrates the effect of TTX on the Na⁺ current expressed in a cell maintained in culture for 7 days. When the cell was depolarized from a holding potential of −80 mV to +20 mV in 10 mV steps, the Na⁺ current was activated with voltage pulses more positive than −40 mV (Fig.4A). In Figure 4B, the same voltage protocol was repeated during superfusion of a medium containing TTX. The Na⁺ current was unaffected by application of 10 μM TTX (Fig. 4B) that would typically be expected to completely block TTX-sensitive Na⁺ channels in a variety of excitable cells (for review, see Hille, 1984). Concentrations higher than 50 μM TTX slightly suppressed the current in some cells (data not shown). Replacement of external Na⁺ with choline abolished this current (Fig. 4C). Application of another Na⁺ channel blocker STX at concentrations of 10 and 50 μM did not block the Na⁺ current (data not shown).

Fig. 4.

Effect of TTX on inward Na⁺ currents. A: Voltage-gated Na⁺ currents in control solution. B: Currents in control solution containing 10 μM TTX. C: Suppression of the inward Na⁺ currents in Na⁺-free solution.

Onset of Na⁺ current expression

As stated above, some of RPE cells attached onto a substrate became flat and started the first mitosis within a week. Figure 5 shows the appearance rate of voltage-gated Na⁺ current in these cells. Culture period was divided into 3 stages (I, !l and ill) on the basis of the morphology of RPE cells. RPE cells in the stage I, like a cell in Figure 1B, were flat in shape. Their nuclei were invisible, because pigment granules did not spread throughout the cytoplasm. None of them exhibited the Na⁺ current at this stage. RPE cells in the stage II, like a cell in Figure 1C, stretched sufficiently and their nuclei became visible, because the pigment granules spreaded throughout the cytoplasm. Ten out of 18 cells (about 56%) examined expressed the Na⁺ current. RPE cells in the stage III included cells during or just after the cell division (Fig. 1D). Six of 12 cells (50%) expressed the Na⁺ current at this stage.

Fig. 5.

Appearance rate of Na⁺ current-expressing cells at various times within the first cell cycle. Culture period was divided into three stages (I, II and III). Cells in stages I, II and III showed morphological characteristics similar to those in Figs. 1B, 1C and 1D, respectively. The ratio of the number of current-exprssed cells to the total number of cells examined were indicated above each bar.

Effect of trichostatin on RPE cells

RPE cells were cultured in the presence of TSA (0.1y∼1.0 μg/ml). TSA has been reported to block the cell cycle of rat fibroblasts (Yoshida and Beppu, 1988). When TSA at a concentration of 0.1 μg/ml was added to the medium at the beginning of cell culture, it arrested the cell division of cultured RPE cells during a 2 week period of observation. After removal of this drug, mitotic cells appeared after 6 days or more. The drug did not inhibit the cell spreading. At higher concentrations, RPE cells tended to exhibit vacuoles in the cytoplasm for a week in culture and some of them detached from the substrate. This fact may imply the deteriolation of the cells.

After 7 days of incubation with TSA (0.1 μg/ml), the membrane properties were measured to determine whether the Na⁺ current was expressed or not. The result is shown in Figure 6A in comparison with that in the control solution. There was no significant difference in the percentages of the Na⁺ current expression in the presence and absence of TSA. That is, in the control solution, 20 of 31 cells (about 64%) expressed the Na⁺ current, while in the presence of TSA, 17 of 31 cells (about 53%) expressed the current. The mean value of the maximum Na⁺ current in the presence of TSA was 122.9±66 pA (mean±SD, n=17). This value was less than half of the value (316±109 pA, n=20) in the control solution (Fig. 6B).

Fig. 6.

Effects of TSA on Na⁺ current expression. A: Comparison of the appearance rates of Na⁺ current-expressed cells in control solution and solution containing 0.1 μg/ml TSA. Membrane currents were recorded from sufficiently streched cells after 7 days in culture. The ratio of the number of current-exprssed cells to the total number of cells examined were indicated above each bar. B: Amount of Na⁺ currents in control solution and solution containing 0.1 μg/ml TSA. The error bars show±SD.