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(The FASEB Journal. 1998;12:1125-1134.)
© 1998 FASEB

Altered regulation of L-type channels by protein kinase C and protein tyrosine kinases as a pathophysiologic effect in retinal degeneration1

Stefan Mergler1, Kirsten Steinhausen, Michael Wiederholt and Olaf Strauss

a Institut für Klinische Physiologie, Universitätsklinikum Benjamin-Franklin der Freien Universität Berlin, 12200 Berlin, Germany


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 REFERENCES
 
The effect of protein tyrosine kinases (PTK) on L-type calcium channels in cultured retinal pigmented epithelium (RPE) from rats with retinal dystrophy was investigated. Barium currents through Bay K 8644 (10-6 M) sensitive L-type channels were measured using the patch-clamp technique. The current density of L-type currents is twice as high and the inactivation time constants are much slower than in cells from nondystrophic control rats. Application of the PTK blockers genistein, lavendustin A, and herbimycin A (all 5x10-6 M) led to an increase of L-type currents. Intracellular application of pp60c-src (30 U/ml) via the patch pipette led to a transient decrease of L-type currents. The protein kinase A (PKA) and PKG blocker H9 (10-6 M) showed no effect on L-type currents. However, the protein kinase C blocker chelerythrine (10-5 M) reduced these currents. Up-regulation of PKC by 10-6 M 4ß-phorbol-12 myristate-13 acetate (PMA) led to a decrease of L-type currents. Additional application of genistein led to a further decrease of these currents. However, intracellular application of pp60c-src in PMA-treated cells led to a transient increase of L-type currents. Investigating the calcium response to bFGF application showed that RPE cells from RCS rats used different pathways than control RPE cells to increase cytosolic free calcium. This different pathway does not involve the activation of L-type channels. The present study with RPE cells from rats with retinal dystrophy shows a changed integration of PTK and PKC in channel regulation. Considering the altered response to bFGF in RCS-RPE cells, this disturbed regulation of L-type channels by tyrosine kinases is involved in the etiology of retinal degeneration in RCS rats.—Mergler, S., Steinhausen, K., Wiederholt, M., Strauss, O. Altered regulation of L-type channels by protein kinase C and protein tyrosine kinases as a pathophysiologic effect in retinal degeneration. FASEB J. 12, 1125–1134 (1998)


Key Words: retinal pigment epithelium • bFGF • calcium channels • retinal pigment epithelium • RCS rats


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 REFERENCES
 
EVEN THOUGH INVESTIGATION of the regulation of ion channels by protein tyrosine kinases (PTK)3 is at the beginning, it is a field with a fast-growing number of publications. Among others, L-type calcium channels have been shown to be regulated by PTK in various tissues (1–6). In these studies, PTK appeared as an activator of L-type calcium channels. In addition, L-type calcium channels represent an integration site for several second-messenger pathways (7, 8). Recently, we demonstrated that PTK is another factor integrated by these ion channels (4). When different protein kinases act simultaneously on L-type calcium channels, they do not simply activate L-type channels in an additive manner, but work together in a more complex way. Several serine/threonine kinases and PTK of the pp60c-src subtype (9) interact in the regulation of calcium channel activity. This has been demonstrated for the regulation of L-type channels by protein kinase A (PKA) and protein kinase C (PKC) or for their regulation by PTK and PKC (3, 4, 7, 10). In the latter case, the activity of PKC determines whether PTK activates or inhibits L-type calcium channel activity (4).

The retinal pigment epithelium (RPE) expresses L-type calcium channels (11–14), and these ion channels are also regulated by PTK and PKC (3, 4, 15). The RPE interacts closely with photoreceptors of the retina to maintain retinal function (16); it transports nutrients from blood to the retina, maintains the ion homeostasis in the subretinal space, and phagocytoses shed photoreceptor outer membranes (16, 17).

To study the integration of PKC and PTK activity by L-type calcium channels under pathophysiological conditions, we investigated the regulation of these calcium channels in RPE cells that display an altered second-messenger metabolism. We used RPE cells from the Royal College of Surgeons (RCS) rats, which suffer from an inherited retinal degeneration caused by a functional defect of the RPE (18). The RPE can bind shed photoreceptor outer membranes (ROS) but is unable to ingest them (17, 19–21). This inability is caused by an altered second-messenger metabolism that fails to activate phagocytosis when photoreceptor membranes bind to receptors on the cell membrane (22, 23). Mainly, the inositol triphosphate/Ca2+-second messsenger system seems to be involved in this altered regulation (23).

The functional defect of the RPE in RCS rats seems to be linked to the availability of basic fibroblast growth factor (bFGF). The earliest events taking place in the postnatal retina of RCS rats are changes in bFGF secretion (24–26). These changes were observed at postnatal day 8, long before first morphological changes in the retina could be observed. In addition, the functional defect of the RCS-RPE, the inability to phagocytose shed photoreceptor outer segments, could be restored by bFGF (27, 28). Therefore, bFGF appears to be a potent factor to attenuate retinal degeneration in RCS rats (29, 30). In RCS rats, of many cytokines only bFGF was able to prevent damage to the retina (31). Since the RPE normally secretes bFGF (1, 33, 34), we postulate that the altered second-messenger metabolism in the RPE of RCS rats (22, 35) changes the autocrine regulation of bFGF secretion.

Since RPE cells from RCS rats express an increased calcium conductance compared to cells from nondystrophic control rats (12), it is likely that the altered second-messenger metabolism leads to changed calcium channel characteristics. The effects of inhibition or activation of protein kinases were measured using the perforated patch technique (36). PTK pp60c-src was applied intracellularly via the patch pipette by using the conventional whole-cell configuration (37).


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 REFERENCES
 
Cell culture
Primary cultures of RPE cells from 6–8 days old RCS rats were established according to the method of Edwards (38). For culturing, sheets of RPE were isolated and collected in Ham's F10 culture medium supplement with 20% fetal calf serum, 10-4 g/ml kanamycin, and 5 x 10-5 g/ml gentamycin. After suspension of the cells by gentle pipetting, the cells were plated in petri dishes equipped with glass coverslips. The cultures were maintained in 5% CO2 at 37°C. The cultures were used after 3–8 days because the highest density of L-type channel currents has been observed during this period (13).

Perforated patch-clamp recordings
Coverslips with RCS-RPE cells were mounted in the stage of an inverted microscope and superfused with a bath solution containing (in mM): 130 NaCl, 3 TEACl, 0.3 CaCl2, 0.6 MgCl2, 14 NaHCO3, 1 Na2HPO4, 33 HEPES, 6 glucose (pH = 7.2 withTris). The bath solution contained 10 mM BaCl2 as charge carrier to measure currents through L-type channels. All drugs were added from dimethyl sulfoxide (DMSO) containing stock solutions. To avoid influence from DMSO on L-type currents, we used DMSO in the bath solution at a concentration of lower than 0.1% [current density with DMSO was 1.53 ± 0.43 pApF-1 (n=5) and without DMSO was 1.83 ± 0.43 pApF-1 (n=5); P<0.1]. Pipettes of borosilicate or soft glass with a resistance of 3–5 M{Omega} were pulled using an Universal-Puller (Zeitz, Augsburg, Germany). Pipettes were filled with pipette solution containing (in mM): 100 CsCl, 10 NaCl, 0.5 CaCl2, 2 MgCl2, 5.5 EGTA-Tris, 10 HEPES (pH 7.2). For perforated patch recordings, the pipette solution contained 120 x 10-6 g/ml nystatin. The osmolarity of the pipette solution was 3 x 10-2 Osmol lower than the osmolarity of the bath solution. Membrane currents were recorded using an EPC-7 (HEKA, Lamprecht, Germany). Electrical stimulation, data storage, and data processing were performed using TIDA (HEKA, Lamprecht, Germany) software in conjunction with an AT-compatible computer. All electrophysiological experiments were performed at room temperature. Membrane capacitance and access resistance were calculated from the capacitance current transient induced by depolarizing voltage step of 30 ms duration and 10 mV amplitude from the holding potential. In the perforated patch configuration, the cells showed an access resistance of 34.2 ± 3.0 M{Omega} (SEM, n=24). The membrane capacitance was 116.9 ± 12.9 pF (SEM, n=24). Access resistance and membrane capacitance were compensated by the patch-clamp amplifier. Since the amplifier operates with feedback circuitries for access resistance compensation, the access resistance was not fully compensated for values lower than 10 M{Omega} in order to avoid cell destruction. Potentials were corrected for liquid junction potentials. Voltage-dependent barium currents were induced by nine depolarizing voltage steps of 50 ms and 10 mV increasing amplitude at a frequency of 1 Hz from a holding potential of -70 mV.

Intracellular application of the protein tyrosine kinase pp60c-src
Koh (39) has described this type of protein tyrosine kinase in retinal pigment epithelium. Protein tyrosine kinases of the pp60c-src-type (9) were used in our previous study (34). Purified recombinant human PTK pp60c-src was stored in a stock solution (3000 U/ml) and added to the intracellular pipette solution at a dilution of 1:1000 just before use. pp60c-src was applied via the patch pipette by using the conventional whole-cell configuration with the same bath and pipette solution according to Wang and Salter (40). To avoid the activation of chloride currents, the bath solution contained 0.5 mM DIDS. The pipette solution contained 4 mM MgATP and 30 U/ml of pp60c-src added from stock solution. For control experiments, pp60c-src was heat inactivated by incubation for 30 min at 95°C. The RCS-RPE cells showed a membrane capacitance of 97.1 ± 9.8 pF in the whole-cell configuration (SEM, n=20) and an access resistance of 26.8 ± 2.6 M{Omega} (SEM, n=20). With heat-inactivated pp60c-src, the L-type currents showed no rundown for 15 min. To show the effect of intracellular application of substances to the cell, the cell was electrically stimulated every 60 s by a voltage step from -70 mV to +10 mV for 50 ms.

Measurement of intracellular Ca2+ with fura-2 (41)
RPE cells were grown in coverslips for 3–8 days and then incubated with serum-free Ham's F10 culture medium for 24 h. Fura2/AM (5x10-6 M) was added to the bath solution for 20 min just before use. Serum-free medium permits intracellular esterases to cleave fura-2AM to fura-2 (42). Patch-clamp fura experiments were done at room temperature in the bath solution described before. The fluorescent indicator dye fura-2 was loaded by diffusion from the bath solution. Extracellular solution changes were made by slow perfusion. Cells were excited at 340 and 380 nm and relative fluorescence registered at 510 nm with the photomultiplier (Hamamatsu). Rapid interchange between the two excitation wavelengths was done using a rotating filter wheel. We used changes in the 340/380 fluorescence ratio as an index of changes in [Ca2+]i and routinely converted ratios into absolute [Ca2+]i. The two fluorescence intensities were filtered at 500 Hz Bessel Lowpass. Current recordings and signals from the photomultiplier were acquired by a computer-based patch-clamp amplifier system (EPC-9).

Materials
Three PTK inhibitors have been used throughout to describe the effects of PTK inhibitors on L-type currents, because no significant different effects on L-type currents were observed in RPE cells from nondystrophic rats (4). Media and cell culture supplements were purchased from GIBCO Life Technologies (Eggenstein, Germany). All chemicals were purchased from Sigma (München, Germany), Research-Biochemicals (Köln, Germany), Serva (Heidelberg, Germany), and Merck (Darmstadt, Germany). pp60c-src was delivered from Upstate Biotechnology (Lake Placid, N.Y.).

Data and statistical analysis
Data were presented as mean ± SEM and analyzed for significance with the paired Student's t test when relevant to experiments with the PTK and PKC inhibitors. The unpaired Student's t test was used in intracellular applications experiments and experiments with the PKC activator 4ß-phorbol-12 myristate-13-acetate (PMA). Data were considered to be significant at P values of less than 0.05. Data presented in the figures show one typical experiment of three to six performed. In current/voltage plots, current amplitudes were plotted against each step potential of the electrical stimulation. In the whole-cell intracellular application experiments, maximal current amplitudes induced by a voltage step from -70 mV to +10 mV were measured every minute.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
REFERENCES
 
Identification of L-type currents in cultured RPE cells from RCS rats
L-type currents were measured under extra- and intracellular potassium-free conditions in primary cultures of RPE cells from RCS rats. Predominantly one type of voltage-dependent inward current was activated when 10 mM barium was present in the bath solution. The currents in RCS-RPE cells showed a current density of 2.42 ± 0.34 pApF-1 (n=7) in the perforated patch configuration under control conditions. The potentials with maximum current amplitudes were between 0 and +20 mV. These currents activated at potentials more positive than -38 ± 5 mV (n=5) ( Fig. 1C). The inactivation time was 297 ± 36 ms in control conditions (n=3, fitted by single exponential fit) (11). In the presence of the L-type channel activator Bay K 8644 (10-6 M), L-type currents significantly increased from control value (100%) to 44 ± 7% (n=5) (recovery: occurred significantly to 108 ± 6% of control; n=5). The influence of the DHP agonist Bay K 8644 on whole-cell currents kinetics has been described by Bechem et al. (43).



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  		Figure 1. Identification of L-type currents in RPE cells from RCS rats. A) Stimulus protocol: the cell was electrically stimulated via the patch pipette using a series of voltage steps from the holding potential at 1 s intervals. From the holding potential (-70 mV), 10 voltage steps of 50 ms duration and 10 mV increasing amplitude were performed to depolarize the cell. B) Currents under control conditions and in the presence of the L-type channel opener Bay K 8644. Currents were corrected for capacitance artifacts and leak currents. Left: In control conditions, electrical stimulation as shown in panel A led to sustained voltage-dependent inward currents. Right: In the presence of 5 10-6 M Bay K 8644 in the bath solution, the voltage-dependent inward currents were increased. C) Effect of Bay K 8644 depicted as currents/voltage plot. The maximal current amplitudes were plotted against the pipette potentials of the stimulation protocol shown in panel A. Application of 5 x 10-6 M Bay K 8644 reversibly increased the amplitudes of the inward currents. Thus, the sustained inwardly directed barium currents were sensitive to the L-type channel opener Bay K 8644.

Effect of inhibition of protein tyrosine kinases on L-type currents
Table 1 lists the effects of protein tyrosine kinase (PTK) inhibitors on L-type calcium currents. In the presence of the specific PTK inhibitor genistein (44) (5x10-6 M), the L-type currents were reversibly increased to 131.3 ± 10.8 (n=6) of control (recovery: 109.2 ± 4.7% of control; n=4) ( Fig. 2A, B ) and the values for the PTK blocker lavendustin A were 136.8 ± 5.0% (n=7) of control (recovery: 112.2±7.4% of control; n=5), respectively. Results for lavendustin A do not appear in Fig. 2.


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  		Table 1. Effects of PTK inhibitors on L-type calcium currents



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  		Figure 2. Effect of protein tyrosine kinases on L-type currents in RPE cells from RCS rats. A) Effect of the PTK blocker genistein (5X10-6 M) on L-type currents at a test potential of +10 mV. B) Current/voltage plot summarizing an experiment with genistein (5X10-6 M). C) Current/voltage plot summarizing an experiment with the irreversibly PTK blocker herbimycin A (5 X 10-6 M). D) Plot of the changes of the peak L-type current amplitudes estimated once every minute after breaking into the whole-cell (t = 0 min) configuration by a voltage step from -70 mV to +10 mV for 50 ms. Peak current amplitudes of 14 cells were normalized to the individual current amplitudes measured 3 min after breaking into whole-cell configuration (current density: 1.77;pm0.39 pA/pF; n = 14). Control currents were measured in the whole-cell configuration with heat-inactivated pp60c-src (n = 5) or without pp60c-src (n = 4) in the patch pipette. With 30 U/ml active pp60c-src (n = 5) in the pipette, a transient decrease of the L-type current amplitudes was observed. E) Current/voltage plot showing current amplitudes 3, 5, and 11 min after breaking into the whole-cell configuration with pp60c-src in the pipette.

Extracellular application of the PTK blocker herbimycin A (5x10-6 M) led to an increase to 128.0 ± 13.8% (n=4) of control (recovery: 105.8±22.6% of control; n=4) ( Fig. 2C). In some cells, currents decreased after washout of herbimycin A, which was due to rundown of L-type currents ( Fig. 2C).

Effect of intracellular application of pp60c-src on L-type currents
Fig. 2). Figure 2D shows the decreasing effect of the normalized L-type current amplitudes evoked by intracellular application of pp60c-src (30 U/ml) to RCS-RPE cells. The cells were clamped at -70 mV by using the conventional whole-cell technique. As control, heat-inactivated pp60c-src led to stable recordings of L-type currents for 12 min. At about 13 min, a rundown of L-type current amplitude was observed. At approximately 6 min, the current amplitudes in RCS-RPE cells with active pp60c-src were significantly lower than those without. Under control conditions (only ATP or heat-inactivated pp60c-src), the L-type current amplitude was 109 ± 5% (n=6) of the current measured directly after breaking into the whole-cell configuration. Six minutes after breaking into the whole-cell configuration with pp60c-src in the pipette, the current amplitude was 67 ± 13% (n=4) of the current measured initially. For comparison, current density decreased from the control value of 1.78 ± 0.48 pApF-1 (n=3) directly after breaking into whole-cell configuration to 0.92 ± 0.26 pApF-1 (n=4) 6 min after breaking into the whole-cell configuration with pp60c-src in the pipette.

Interaction of PTK inhibition and the activity of serine/threonine protein kinases
Extracellular application of the PKA and protein kinase G (PKG) blocker H9 (10-6 M) did not lead to significant changes in L-type peak currents (with 86.8±5.7% of control, n=10; recovery: 82.2 ± 8.4 of control, n=5). In the presence of H9, genistein (5x10-6 M) was able to reversibly increase the maximal current amplitude to 134.0 ± 22.2% (n=8) of control; recovery occurred to 82.2 ± 8.5% (n=5) of control (results for H9 were not shown in the figures). Chelerythrine, a PKC blocker, had a decreasing effect on L-type currents to 77.3 ± 10.6% (n=5) at a concentration of 10-5 M; recovery occurred to 89.8 ± 4.6% (n=4). However, in the presence of chelerythrine, lavendustin A (5x10-6 M) increased L-type current amplitudes to 147.2 ± 13.3% (n=5); recovery occurred to 96.7 ± 3.3 (n=3) ( Fig. 3).A–B). Table 1lists the effects of inhibition of PKC and protein tyrosine kinases on L-type calcium currents.



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  		Figure 3. Interaction of protein tyrosine kinase inhibition and protein kinase C activity on L-type currents in RPE cells from RCS rats. A) Maximal current amplitudes induced by a voltage step from -70 mV to +10 mV are depicted in percent of control values before application of the PKC blocker chelerythrine (10-5 M) and the PTK blocker lavendustin A (5X10-6 M). B) Current/voltage plot summarizing an experiment with lavendustin A (5X10-6 M) in the presence of chelerythrine (10-5 M). C) Effect of genistein (5 X 10 -6 M) on L-type currents at a test potential of +10 mV in RCS-RPE cells preincubated in PMA (10-6 M) for 30 min. D) Current/voltage plot summarizing an experiment with genistein (5 X 10 -6 M) preincubated in PMA. E) Maximal current amplitudes induced by a voltage step from -70 mV to +10 mV are depicted in percent of control values before application of genistein (5 X 10 -6 M). F) Summary of the experiments with PKC inhibition or activation in combination with PTK inhibition with normalized peak currents in percent to control before drug application (recoveries were observed in every cell after washout of the blocker).

In the next series of experiments, we attempted to determine whether up-regulation of PKC led to different effects of PTK on L-type currents. Figure 3C–E summarizes the results of these experiments. Indeed, the increasing effect of the specific PTK blocker genistein (44) becomes inverse ( Fig. 1F). First, up-regulation of PKC by incubation of the cells for 30 min in 10-6 M PMA before patch-clamp recording led to a significant reduction of the current density to 0.84 ± 0.12 pApF-1 (n=4) in the perforated patch configuration. In untreated RCS-RPE cells, the current density was 2.42 ± 0.34 pApF-1 (n=7). This value is higher than in RPE cells from nondystrophic rats, which is due to the increased calcium conductance in RCS-RPE cells (11–13). Second, as summarized in Fig 3F, the effect of genistein was reversed in RCS-RPE cells treated with PMA. Now genistein (5x10&?minus;6 M) led to a decrease of the maximal current amplitude ( Fig. 3E) to 30.6 ± 7.1% (n=5); recovery occurred to 74.0 ± 14.2% (n=4). Table 1lists the effect on L-type calcium currents relevant to interaction of PTK inhibition and the activity of serine/threonine protein kinases.

Effect of intracellularly applied protein tyrosine kinase in the presence of up-regulated PKC
Finally, we examined the effect of intracellular application of PTK on L-type currents in PMA treated RCS-RPE cells. As shown in Fig. 4B, in PMA-treated RCS-RPE cells, PTK pp60c-src led to a transient increase of the maximal current amplitudes. The maximal effect was observed 6 min after breaking into the whole-cell configuration. After this time, currents were 138 ± 15% (n=6) of the initial values. Application of heat-inactivated pp60c-src (30 U/ml) via the patch pipette or without application of pp60c-src; current amplitudes were 90 ± 3% (n=5) of the current amplitudes measured directly after breaking into the whole-cell configuration. For comparison, current density in PMA-treated RCS-RPE cells with pp60c-src increased from control value of 1.83 ± 0.50 pApF-1 (n=5) directly after breaking into whole-cell configuration to 4.09 ± 2.17 pApF-1 (n=5) 6 min after breaking into the whole-cell configuration with pp60c-src in the pipette.



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  		Figure 4. Effect of intracellular application of tyrosine kinase on L-type currents in PMA-treated RPE cells from RCS rats. A) Current/voltage plot showing current amplitudes 3 and 5 min after breaking into the whole-cell configuration with pp60c-src in the pipette. RCS-PPE cells were preincubated in PMA (10-6 M). B) Plot of changes in peak L-type current amplitudes estimated once a minute after breaking into the whole-cell (t=0 min) configuration by a voltage step from -70 mV to +10 mV for 50 ms. Peak current amplitudes of 15 cells preincubated in PMA (10-6 M) were normalized to the individual current amplitudes measured 3 min after breaking into whole-cell configuration (current density: 1.86±0.38 pA/pF; n=15). Control currents were measured in the whole-cell configuration with heat-inactivated pp60c-src (n=5) or without pp60c-src (n=4) in the patch pipette. With 30 U/ml active pp60c-src (n=6) in the pipette, a transient increase of the L-type current amplitudes was observed.

Effect of bFGF on [Ca2+]i
To elucidate the influence of the changed regulation of L-type Ca2+ channels on bFGF-induced intracellular Ca2+ transients, cytosolic free Ca2+ ([Ca2+]i) was measured using the fluorescence dye fura-2 as a Ca2+ indicator. RPE cells from control rats and RCS rats display no differences in the basic Ca2+ concentration, which was 111 ± 19 nM (n=17). Before application of bFGF, the cells were held under serum-free conditions for 24 h. In cells from both rat strains, extracellular application of bFGF (10 ng/ml) led to a monophasic slow rise in the intracellular Ca2+, which usually takes 10–15 min to reach steady-state Ca2+ levels ( Fig. 5). The bFGF-induced steady-state Ca2+ concentration was 571 ± 194 nM (n=4) in cells from control rats and 562 ± 146 nM (n=4) in cells from RCS rats. No significant difference exists between these values. In a subsequent series of experiments, we investigated the effect of various substances on the elevation of internal Ca2+ by bFGF ( Fig. 5). In these experiments, the level of [Ca2+]i 3 min after application of bFGF was set as 100%. A second [Ca2+]i value was obtained 5 min after the first and expressed in percent of the first value. Various drugs were tested during the interval between these two times. Under control conditions (no drug given between the third and eighth minute after application of bFGF), the fluorescence ratio rose to 238 ± 24% (n=6) of the initial value in RPE cells from both rat strains. In nondystrophic RPE cells, extracellular application of nifedipine (10-6 M) reduced (n=2) or attenuated (n=2) the bFGF-induced rise in intracellular Ca2+. After a 5 min application of nifedipine, the bFGF-induced Ca2+-level was 97.4 ± 25% (n=4) of the level before application of nifedipine. In comparison, when the same experimental procedure was carried out using RPE cells from RCS rats, [Ca2+]i levels rose to 210 ± 19% (n=4) of the original value, so that in this case nifedipine did not affect the bFGF-induced rise in [Ca2+]i. The bFGF-induced rise in [Ca2+]i was also sensitive to genistein (5x10-6 M). In cells from control rats, application of this tyrosine kinase inhibitor attenuated (n=2) or reduced (n=2) the bFGF-induced rise in [Ca2+]i (5 min value was 99.6±10% of initial value, n=4). In contrast, in RPE cells from dystrophic rats, genistein (5x10-6 M) not only failed to reduce the bFGF-induced rise in [Ca2+]i, but led to a marked increase to 502 ± 64% (n=5) of the initial value, doubling the rise without genistein.



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  		Figure 5. Effect of bFGF (10 ng/ml) on cytosolic free Ca2+ in RPE cells from RCS and control rats. A) Effect of nifedipine (10-6 M) on the bFGF-induced rise in cytosolic free Ca2+ in RPE cells from control rats. Changes in cytosolic free Ca2+ are depicted as the ratio of the fluorescence induced by the excitation wavelengths 340 and 380 nm. B) Effect of genistein (5x10-6 M) on the bFGF-induced rise in cytosolic free Ca2+ in cells from control rats. Changes in cytosolic free Ca2+ are depicted as the ratio of the fluorescence values induced by the excitation wavelengths 340 and 380 nm. C) Effect of genistein (5x10-6 M) on the bFGF-induced rise in intracellular free Ca2+ in RPE cells from RCS rats. Changes in cytosolic free Ca2+ are depicted as the ratio of the fluorescence induced by the excitation wavelengths 340 and 380 nm. D) Comparison of the effects of nifedipine (10-6 M) and genistein (5x10-6 M) on the bFGF-induced rise in intracellular Ca2+. The intracellular Ca2+ level after a 3 min application of bFGF (10 ng/ml) was set as 100%, which was used to normalize the cytosolic Ca2+ 5 min later. t = 8 min corresponds to the Ca2+ level without blocker. E) Comparison of the nifedipine and the genistein effects between control and RCS rat using the data shown in panel D.

DISCUSSION
L-type calcium channels represent an integration site of different second-messenger pathways. In a recent study, we showed that the activity of PKC determines the effect of protein tyrosine kinase on L-type calcium channels (4). In the present study, we investigated the interaction of PKC and PTK under pathophysiologic conditions. RPE cells from RCS rats, which are known to exhibit an altered second-messenger system (23), display changed ion channel characteristics of L-type calcium channels compared to normal RPE cells. These alterations are due to altered regulation of L-type channels. Unlike in other tissues, protein tyrosine kinase inhibits L-type calcium channels in RPE cells from rats with retinal dystrophy and PKC determines the effect of PTK in an inverse mode. We suggest that an altered integration of PTK and PKC activity is involved in the generation of a functional defect, which leads to retinal dystrophy. The results of all experiments relevant to the effects of PTK and PKC on L-type calcium current density are summarized in Table 1. These results are compared to results from a previous study in which we performed the same experiments with RPE cells from nondystrophic control rats (4, 11, 12, 15).

Like RPE cells from human or nondystrophic control rats (3, 4), RPE cells from RCS rats express L-type calcium channels (11–13). In these cells, barium currents induced by depolarization activated at potentials more positive than -38 mV and reached maximal current amplitudes at +10 mV. These currents showed inactivation time constants slower than 100 ms and could be enhanced by the dihydropyridine compound Bay K 8644. However, the currents observed in RPE cells from rats with retinal dystrophy were different from those observed in the nondystrophic variety. The current density of L-type currents in RPE cells from rats with retinal dystrophy is twice as high and the inactivation time constants are much slower than in RPE cells from control rats. This is the explanation for the increased calcium conductance in RPE cells from dystrophic rats, which we have published earlier (12). The reason for these differences seems to be the regulation of L-type calcium channels by PTK and PKC.

L-type calcium channels are regulated by PTK in RPE cells from RCS rats. The barium currents could be enhanced by the PTK blockers lavendustin A (45), herbimycin A (46, 47), and genistein (44). In contrast to the reversible blockers genistein and lavendustin A, we did not observe a recovery with herbimycin A, which is an irreversible PTK blocker. Furthermore, intracellular application of PTK subtype pp60c-src via the patch pipette led to a transient decrease of L-type currents in RPE cells from RCS rats. Since two chemically different compounds enhanced the currents and PTK itself decreased L-type currents, we suggest that PTK led to inhibition of L-type calcium channels in RPE cells from rats with retinal dystrophy. This observation is in contrast to the effect of PTK on L-type channels in other tissues in healthy subjects such as human RPE cells or RPE cells from control rats. In cardiomyocytes and vascular smooth muscle cells, PTK activates L-type calcium channels (2, 5, 6), as has been observed in human RPE cells or RPE cells from control rats (3, 4).

L-type calcium channels in RPE cells from RCS rats are also regulated by serine/threonine kinase. In the present study, the compound H9 (10-6 M) (48) did not alter L-type currents. In contrast, PKC activated L-type channels in RPE cells from rats with retinal dystrophy. This has been shown when using the PKC specific blocker chelerythrine (49). At first glance, regulation of L-type calcium channels by PKC-RPE cells from rats with retinal dystrophy seems to be comparable to RPE cells from control rats. However, there are differences in the regulation by PKC. In RPE cells from rats with retinal dystrophy, activation of PKC by incubation in phorbolesters (50) led to a reduction of L-type currents, whereas no effect of phorbolesters was observed in RPE cells from control rats. Since activation of PKC or inhibition of PKC showed the same effect on L-type currents in RPE cells from rats with retinal dystrophy, we suggest that PKC seems to influence L-type calcium channels by different pathways (in the resting cell for ‘housekeeping’ function and as part of a signal transduction cascade). The difference in RPE cells from control rats may be due to the altered protein phosphorylation in general in RPE cells from RCS rats (51). However, the reason for this needs clarification.

As has been shown in human RPE cells and RPE cells from nondystrophic rats or smooth muscle cells, protein kinases seem to interact in the regulation of L-type channels (3, 4, 7). Thus, these ion channels represent an integration site of different second-messenger pathways. Since the effect of PTK on calcium channels in RPE cells from RCS rats was unchanged in the presence of PKA/PKG blocker H9, we conclude that these serine/threonine kinases do not seem to interact with PTK. In contrast, PKC and PTK interact in the regulation of L-type calcium channels. Inhibition of PKC led to a reduction of L-type currents and the additional inhibition of PTK in the presence of a PKC blocker led to an increase of the current as in control conditions. However, when PKC was activated by incubation of RPE cells in phorbolesters, PTK led to an increase of L-type currents. This was demonstrated by intracellular application of PTK and by inhibition of PTK by lavendustin in cells treated with PMA. As in RPE cells from control rats (4), the activity of PKC also determines the effect of PTK on the L-type calcium channel in RPE cells from rats with retinal dystrophy. In RPE cells from rats with retinal dystrophy, this interaction of PTK and PKC is in an inverse mode compared to the interaction in RPE cells from nondystrophic control rats. In RPE cells from nondystrophic controls, PTK activates L-type calcium channels with unstimulated PKC and PTK inhibits L-type channels when PKC is up-regulated by phorbolesters. Thus, in cells with a disturbed second-messenger metabolism, the integration of different second-messenger pathways by L-type calcium channels is changed.

The hereditary retinal dystrophy in RCS rats is due to a functional defect in the retinal pigment epithelium. The RPE is unable to phagocytose shed ROS (17, 20, 21). The phagocytosis of photoreceptor outer segments by the normal RPE is regulated by the inositol-triphosphate/Ca2+ second-messenger system (52, 53). In addition, a recent study demonstrated that phagocytosis of photoreceptor outer membranes is activated by PTK (54).

L-type Ca2+ channels are involved in the IP3/Ca2+ second-messenger system via regulation by tyrosine kinase. This was shown in RPE cells from nondystrophic rats by attenuation or decrease of the bFGF-induced rise in the cytosolic free Ca2+ by the L-type channel blocker nifedipine and by the tyrosine kinase inhibitor genistein. Thus, in RPE cells from control rats, bFGF led to an influx of extracellular Ca2+ via L-type Ca2+ channels into the cell. Since application of nifedipine could only attenuate and not fully block the bFGF-induced rise in cytosolic free Ca2+, there must be second pathway enabling an influx of extracellular Ca2+ into the cell (55). Genistein and nifedipine were similarly effective. It appears that the involvement of L-type channels in the bFGF response occurs via activation of these ion channels by tyrosine kinase. In RPE cells from RCS rats, application of nifedipine had no effect on the bFGF-induced rise in the cytosolic free Ca2+. Thus, in cells from RCS rats, stimulation of the cytosolic free Ca2+ is enabled via the alternative pathway. This could possibly involve other Ca2+ release-activated channels (55). Application of the tyrosine kinase inhibitor genistein to RPE cells from RCS rats led to an acceleration of the bFGF-induced rise in intracellular free Ca2+. Thus, as is to be expected from the regulation of L-type channels by protein tyrosine kinase in RCS-RPE, inhibition of tyrosine kinase led to the amplification of the bFGF-induced rise in intracellular Ca2+. We conclude that, in RPE cells from RCS rats, the cell must use an alternative pathway to produce a Ca2+ response induced by bFGF because the regular pathway via tyrosine kinase-dependent activation of L-type channels is not available.

In secreting cells, L-type channels play a key role in regulation of the release of hormones or growth factors (55). We propose that tyrosine kinase-dependent activation of L-type Ca2+ channels may be involved in the autocrine regulation of cytokine secretion by RPE cells. Since RPE cells in RCS rats are not able to activate L-type channels via tyrosine kinase and therefore unable to response to bFGF via influx of Ca2+ into the cell via L-type channels, we conclude the autocrine regulation of bFGF secretion is disturbed in these cells in the form of disturbed feedback mechanisms. In many investigations of the hereditary retinal degeneration in RCS rats, bFGF appeared to play a key role in the pathophysiologic processes in the retina. The earliest changes in the RCS retina are changes in the bFGF secretion (26) at postnatal day 8. This is long before the first morphological changes occur (56). Cultured RCS-RPE cells showed a deficiency of bFGF receptor or expression (57). The main pathophysiological process in the RCS retina is the inability to phagocytose shed photoreceptor outer segments by the RPE (17, 20, 21, 58). This functional defect could be restored by bFGF (27, 28). In addition, bFGF appeared as the only factor able to reduce retinal degeneration in RCS rats (29–31). We have shown (to our knowledge, for the first time) that a changed regulation of L-type channels by tyrosine kinase is involved in a pathophysiologic process.


   ACKNOWLEDGMENTS
 
This work was supported by the DFG grant Wi 993/1–3, Str 480/3–1, and Deutsche Retinitis Pigmentosa Patientenvereinigung. The technical assistance of A. Krolik and M. Boxberger are gratefullly acknowledged. The authors also want to thank Dr. F. Stumpff for helpful discussions.


   FOOTNOTES
 
1 Parts of this work have been published in abstract form [S. Mergler et al. (1996) Pflueger's Arch. 431 (Suppl.), R114].

1 Correspondence: Institut für Klinische Physiologie, Universitätsklinikum Benjamin-Franklin, Freie Universität Berlin, Hindenburgdamm 30, 12200 Berlin, Germany. E-mail: wiederho{at}zedat.fu-berlin.de

3 Abbreviations: pp60c-src, type of protein tyrosine kinase; bFGF, basic fibroblast growth factor; DIDS, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid; EGTA, ethylene glycol-bis(ß-aminoethyl ether) N,N,N',N'-tetraacetic acid; HEPES, N-[hydroxyethyl] piperazine-N'-[2-ethansulfonic acid]; TEACl, tetraethylammonium chloride; Tris, tris(hydroxymethyl)-amino methane; DMSO, dimethyl sulfoxide; PMA, 4ß-phorbol-12 myristate-13 acetate; RPE, retinal pigment epithelium; PTK, protein tyrosine kinases; PKA, protein kinase A; RCS, Royal College of Surgeons; ROS, photoreceptor outer membranes.

Received for publication September 8, 1997. Accepted for publication April 6, 1998.


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