Inside bluegill (Lepomis macrochirus) retinal pigment epithelial cells, pigment granules move in response to extracellular signals. During the process of aggregation, pigment motility is directed toward the cell nucleus; in dispersion, pigment is directed away from the nucleus and into long apical processes. A number of different chemicals have been found to initiate dispersion, and carbachol (an acetylcholine analog) is one example. Previous research indicates that the carbachol-receptor interaction activates a Gq-mediated pathway which is commonly linked to Ca2+ mobilization. The purpose of the present study was to test for involvement of calcium and to probe calcium-dependent mediators to reveal their role in carbachol-mediated dispersion.
Carbachol-induced pigment granule dispersion was blocked by the calcium chelator BAPTA. In contrast, the calcium channel antagonist verapamil, and incubation in Ca2+-free medium failed to block carbachol-induced dispersion. The calcineurin inhibitor cypermethrin blocked carbachol-induced dispersion; whereas, two protein kinase C inhibitors (staurosporine and bisindolylmaleimide II) failed to block carbachol-induced dispersion, and the protein kinase C activator phorbol 12-myristate 13-acetate failed to elicit dispersion.
A rise in intracellular calcium is necessary for carbachol-induced dispersion; however, the Ca2+ requirement is not dependent on extracellular sources, implying that intracellular stores are sufficient to enable pigment granule dispersion to occur. Calcineurin is a likely Ca2+-dependent mediator involved in the signal cascade. Although the pathway leads to the generation of diacylglycerol and calcium (both required for the activation of certain PKC isoforms), our evidence does not support a significant role for PKC.
Organelle motility is an essential function of all cells. The shuttling of supramolecular structures is regulated by motor proteins, cytoskeletal elements, and a wide variety of chemical messengers. Pigment cells are an excellent model in which to study cell motility because pigment granules are readily visible, move rapidly, and undergo reversible movements which can be manipulated experimentally . Found in a variety of cell types, pigment granule motility in the retinal pigment epithelium (RPE) was examined in the present study.
The RPE is a single layer of cells found between the neural retina and the choroid. In animals that do not possess the ability to constrict the pupil, RPE cells possess apical processes which interdigitate with photoreceptors [2, 3]. Within each cell, pigment granules aggregate and disperse. In the aggregated state, pigment granules are withdrawn from the apical processes and cluster in the cell body (Figure 1A–B), while in the dispersed state, they are moved down the lengths of apical processes as shown in Figure 1C–D. In the dispersed state, protection of rod photoreceptors from photobleaching is thought to be enhanced [3–6].
Extracellular molecular mediators stimulate pigment granule motility, and several different agents have been identified that induce movement. Forskolin (FSK) stimulates adenylyl cyclase to increase intracellular levels of cAMP, resulting in aggregation [7–14]. Catecholamines and their agonists (epinephrine, phenylephrine, clonidine, apomorphine, and dopamine) induce dispersion [9, 13, 15]. Dopamine acts through D2 receptors which inhibit adenylyl cyclase . With adenylyl cyclase inhibited, [cAMP]i decreases and dispersion ensues.
Catecholamines are not the only extracellular messengers that induce pigment granule dispersion in RPE. In 1998, García  reported that the acetylcholine analog carbachol induces pigment granule dispersion in RPE isolated from green sunfish (Lepomis cyanellus). González et al.  extended this finding to RPE isolated from bluegill (Lepomis macrochirus) and further reported that muscarinic Modd receptor activation leads to pigment granule motility. Later it was found that the native ligand acetylcholine induces pigment granule dispersion . Following Modd receptor activation, phospholipase C is activated, cleaving PIP2 to generate diacylglycerol and inositol trisphosphate (IP3). Antagonists to the IP3 receptor inhibited carbachol-induced dispersion .
In other systems, the IP3 receptor has been found within the membrane of the endoplasmic reticulum. With ligand bound to the IP3 receptor, Ca2+ stored within the ER lumen is released into the cytosol (see ). Extrapolating these observations to regulation of pigment granule movement in RPE, one might infer a role for Ca2+ in regulating pigment granule dispersion in RPE. However, King-Smith et al.  were unable to demonstrate a role for Ca2+ in either pigment granule dispersion or aggregation. Rather, they found that when dispersion is experimentally induced by cAMP washout in RPE isolated from green sunfish (Lepomis cyanellus), there is no significant rise in intracellular Ca2+ levels nor does chelating Ca2+ prevent pigment granule dispersion. This finding may not, however, rule out a role for Ca2+ in a physiological setting involving receptor activation if the physiological role for Ca2+ precedes a decrease in intracellular [cAMP]. Therefore, the hypothesis driving our study was that Ca2+ is required for carbachol-induced dispersion in RPE cells isolated from bluegill, which we examined using calcium chelators, calcium channel blockers and calcium-free media. Furthermore, we predicted that if calcium were required, calcium-dependent effectors would also be expected to play a role in pigment granule dispersion; therefore, intracellular mediators involved in pigment granule dispersion were explored.
Exploration for calcium requirements in carbachol-induced pigment granule dispersion
To test whether an elevation in intracellular Ca2+ was required for carbachol-induced pigment granule dispersion, RPE cells were isolated, treated with forskolin (FSK) and then challenged to disperse in the presence or absence of the membrane-permeant Ca2+ chelator BAPTA-AM. When cells were incubated with 100 nM carbachol in the presence of 1 μM BAPTA-AM or 30 μM BAPTA-AM, the resulting pigment index (PI) values (0.63 ± 0.02, n = 3; 0.66 ± 0.02, n = 3, respectively) were not statistically significantly different from the PI of cells treated with FSK (0.62 ± 0.01, n = 54) but were different from PI of cells treated with carbachol alone (PI = 0.89 ± 0.01, n = 54) (Figure 2). The maximum concentration of DMSO used during incubation (5% vol/vol) did not alter PI values in controls (PI carbachol = 0.89 ± 0.01, n = 54; PI carbachol in 5% DMSO = 0.86 ± 0.00, n = 3).
To test the importance of extracellular Ca2+ in carbachol-induced pigment granule dispersion, verapamil, an L-type Ca2+ channel antagonist, was tested for its ability to block carbachol-induced dispersion. Verapamil failed to block carbachol-induced dispersion at both 10 μM (PI = 0.84 ± 0.04, n = 3) and 100 μM (0.89 ± 0.01, n = 3) concentrations. The difference in PI between cells with and without the Ca2+ channel blocker was not statistically significant (Figure 2). In addition, the differences between the pigment indices for either condition (carbachol or carbachol plus verapamil) and the FSK condition were statistically significant.
To confirm that an influx of Ca2+ from the extracellular medium was not required, RPE cells were isolated, treated with forskolin (FSK) and then challenged to disperse in the presence or absence of extracellular Ca2+. RPE were treated with 10 μM FSK to induce pigment granule aggregation followed by carbachol (100 nM) to induce pigment granules to disperse. The difference in PI between cells with (0.89 ± 0.01, n = 54) and without (0.86 ± 0.03, n = 6) extracellular Ca2+ available was not statistically significant (Figure 2). However, the difference between either condition (with and without Ca2+) and the 10 μM FSK condition was statistically significantly different.
Investigation of calcium-dependent mediator proteins
Since our results using BAPTA-AM suggested a role for intracellular Ca2+, we tested possible downstream, Ca2+-dependent mediators. We first tested whether protein kinase C (PKC) is required for carbachol-induced dispersion by using two inhibitors (staurosporine and bisindolylmaleimide II). Cells treated with carbachol in the absence or presence of staurosporine (100 nM, 0.83 ± 0.04, n = 4; 500 nM, 0.88 ± 0.01, n = 3) were found to differ from the cells treated with FSK, but were not different from each other (Figure 3). When bisindolylmaleimide II (20 nM, 0.89 ± 0.03, n = 3; 200 nM, 0.80 ± 0.03, n = 6; 2 μM, 0.88 ± 0.01, n = 3) was applied with carbachol, dispersion was partially inhibited, but only in the 200 nM treatment (Figure 3). Exhibiting a 20% inhibition in dispersion, the cells treated with 200 nM bisindolylmaleimide II were statistically significantly different from both FSK and carbachol treatments.
To test whether activating PKC was sufficient to induce dispersion, PMA was applied to RPE. Following FSK treatment, PMA (without carbachol) elicited PI values (1 μM, 0.67 ± 0.01, n = 3; 10 μM, 0.64 ± 0.03, n = 3; 100 μM, 0.67 ± 0.02, n = 3) that were not significantly different from FSK-treated cells (Figure 4).
Another Ca2+-dependent effector tested was the protein phosphatase calcineurin. The calcineurin inhibitor cypermethrin (100 pM) completely inhibited carbachol-induced pigment granule dispersion (Figure 5). The cypermethrin condition (0.62 ± 0.06, n = 3) was not significantly different from the FSK condition (0.62 ± 0.01, n = 54), but was different from the carbachol condition (0.89 ± 0.01, n = 54).
The results indicate that an elevation of intracellular Ca2+ is required for carbachol-induced pigment granule dispersion. Furthermore, the necessary Ca2+ may be derived entirely from intracellular stores, and one effector activated by Ca2+ is the protein phosphatase calcineurin. In contrast, our results using inhibitors and activators of PKC suggest that the activity of this enzyme is neither necessary for carbachol-induced pigment granule dispersion nor sufficient to drive pigment granule dispersion.
Carbachol-mediated dispersion requires activation of the IP3 receptor , suggesting that intracellular Ca2+ stores are accessed following muscarinic receptor activation . Chelating intracellular Ca2+ with BAPTA completely blocks carbachol-induced dispersion. That this Ca2+ is most likely coming from intracellular stores (regulated by the IP3 receptor) and not from the extracellular environment is supported by the observations that carbachol-induced dispersion is blocked by the IP3 receptor antagonist 2-aminoethoxydiphenyl borate  and eliminating extracellular Ca2+ or blocking its entry across the plasma membrane failed to inhibit dispersion. There are many Ca2+-requiring mediators that could be involved in carbachol-induced dispersion, including the Ca2+-calmodulin-dependent protein phosphatase calcineurin. Since cypermethrin inhibits calcineurin activity, finding that cypermethrin blocks carbachol-induced pigment granule dispersion suggests that calcineurin is involved. This result correlates with work done by Thaler and Haimo . Studying melanophores, they found a role for calcineurin in pigment aggregation. Furthermore, García  found that okadaic acid induces aggregation in RPE cells of green sunfish, suggesting a role for protein phosphatases in maintaining pigment granule dispersion.
One possible role for calcineurin is to remove phosphate groups added to proteins by PKA . When FSK is applied to cells, cAMP levels increase , leading to PKA activation  and eventual aggregation of pigment granules. In the RPE pigment motility system, the substrate phosphorylated by PKA is as yet unknown. Prior to dispersion, cAMP levels drop and likewise PKA activity. Increased Ca2+ concentrations inside the cell may lead to calcineurin activation that may remove phosphate groups from substrates of PKA, enabling pigment granule dispersion to occur. Our finding that cypermethrin inhibits carbachol-induced dispersion is consistent with this model.
As stated before, inhibition of PLC blocks carbachol-induced dispersion . Since PLC cleaves PIP2 into IP3 (leading to Ca2+ mobilization) and DAG, and DAG and Ca2+ together activate protein kinase C, protein kinase C seemed likely to be a mediator of carbachol-induced dispersion . This hypothesis seemed the more likely since in some species, pigment dispersion in melanophores appears to be regulated by PKC [25, 26]. However, our results from three separate experiments failed to support a role for PKC in carbachol-induced dispersion. Treatment with the PKC activator PMA did not induce pigment granule dispersion. Neither of the PKC inhibitors tested (bisindolylmaleimide and staurosporine) inhibited carbachol-induced pigment granule dispersion at most concentrations tested. Cells treated with bisindolylmaleimide II at 200 nM experienced a 20% inhibition in dispersion relative to the carbachol-treated cells when data from the latter were compiled from all experiments. However, when comparison was made only to the carbachol-treated cells from the experiment in which the 200 nM concentration was tested, the difference in mean pigment indices was not found to be statistically significant. This case was the only one in which the compilation of the data yielded a statistical outcome different from analysis of the data within single experiments. Nevertheless, it remains possible that PKC has a role in carbachol-mediated dispersion which parallels the main pathway linking receptor activation to pigment granule movement. It should be mentioned that definitive evidence that PKC is expressed in bluegill RPE has yet to be found. We have found that PKC-α and PKC-β were expressed in bluegill brain, but not in bluegill RPE (unpublished results). These findings are consistent with work reported by others. In the retina of zebrafish, PKC labelling is observed in the neural retina, but not in the retinal pigment epithelium .
Although much remains to be discovered, our results have increased our understanding of the pathway from carbachol to pigment granule dispersion. We propose the following model: First, carbachol binds an Modd muscarinic receptor leading to PLC activation [17, 18]. PLC then cleaves PIP2 into DAG and IP3. A role for DAG in pigment movement in RPE has not been revealed. In contrast, IP3 binds its receptor, thus releasing Ca2+ from intracellular stores . Although the conclusion requires support from Ca2+ imaging, our data indicates that Ca2+ released from such stores is sufficient for downstream mediator activation; in other words, extracellular Ca2+ is not required. Increasing cytosolic Ca2+ leads to calcineurin activation. Calcineurin removes phosphate groups added by PKA on an as yet unidentified protein which functions as a molecular switch regulating the pigment granule position in the retinal pigment epithelium. Refer to Figure 6 for the proposed carbachol-induced dispersion pathway.
Our results indicated that in the RPE of bluegill, carbachol-induced pigment granule dispersion is a process that requires intracellular, but not extracellular, Ca2+. Our evidence also supports a pigment dispersion model that requires calcineurin but not protein kinase C.
The experimental animals were maintained as described previously . All experiments were performed following protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Texas State University- San Marcos (Protocol # 069744F82). Bluegill (Lepomis macrochirus) were purchased from Johnson Lake Management, San Marcos, TX. All fish were acclimated at least two weeks prior to experimentation in indoor, aerated aquaria on a 12 hour: 12 hour (light/dark) cycle.
To extract RPE, fish were captured 6 hours into the light cycle and dark adapted in a room void of light. In the dark room, the fish were allotted thirty minutes to adjust to the darkness in aerated aquaria. After thirty minutes, the spinal cord was severed, and the fish were double-pithed under dim, incandescent light (≤ 2 lux). Light was measured using a Lutron LX-101 lux meter (Lutron, Coopersburg, PA). The eyes were then removed and hemisected along the equatorial axis. The anterior portion of the eye was discarded. The retina was removed from the eyecup, and the RPE was flushed out using bicarbonate-buffered Ringer prepared the day of the experiment . All chemicals were purchased from Sigma-Aldrich, St. Louis, MO. The Ringer's solution contained 24 mM NaHCO3, 3 mM HEPES (free acid), 116 mM NaCl, 5 mM KCl, 1 mM NaH2PO4·H2O, 26 mM dextrose, 1 mM ascorbic acid, 1.12 mM MgSO4, 1 mM EGTA, and 1.8 mM CaCl2, titrated to pH of 7.4 using 1 M NaOH. The buffer was gassed with 95% air/5% CO2 for at least 15' prior to the dissection and throughout the experiment to maintain a pH of 7.2. For one experiment, Ca2+-free Ringer was prepared as above with the omission of CaCl2.
Once the RPE had been isolated, excess Ringer was removed, and forskolin (10 μM) was applied to induce aggregation. The cells were then incubated for 45' in a humidified chamber gassed with a mixture of 95% air and 5% CO2on a gyratory shaker (50 rpm) as in González et al. .
After incubation in forskolin, approximately one third of the collected RPE was fixed using a 2× stock solution of fixative prepared in phosphate buffered saline (PBS). PBS was prepared as 137 mM NaCl, 2.7 mM KCl, 4.3 mM NaH2PO4·H2O, and 1.4 mM KH2PO4 in purified water (resistivity 18 Mohms). Purified water was obtained from a NANOpure Infinity Laboratory Water System. After dilution with an equal volume of Ringer's solution containing tissue, the final concentration of fixative was 0.5% glutaraldehyde, 0.5% paraformaldehyde, and 0.8% potassium ferricyanide.
The remaining cells were divided between two weigh boats and washed clean of forskolin using Ringer. Ringer's solution was pipetted into and out of the weigh boats; about 3 volumes were exchanged. Tissue pieces for both treatment groups were then placed in microcentrifuge tubes containing either 50 μl of 1 μM carbachol and 450 μl of Ringer's solution or 50 μl of 1 μM carbachol, 400 μl of Ringer's solution, and 50 μl of 10× experimental drug (see Pharmacological Agents below). Following an additional 45' incubation, the remaining samples were fixed as described above.
To study the requirement for extracellular and intracellular Ca2+, verapamil and BAPTA-AM (both of which were prepared fresh the day of experimentation) were used, respectively. Cypermethrin (from 1 nM stock solution) was used to inhibit calcineurin (PP2B). Staurosporine, bisindolylmaleimide II, and phorbol esters were used to investigate a role for PKC activity. Staurosporine (Sigma) was divided into 10 μM aliquots in DMSO. Bisindolylmaleimide II (Sigma) was dissolved in DMSO to make a 1 mM stock solution, which was then divided into aliquots and frozen. Phorbol 12-myristate 13-acetate (Sigma) was prepared as a 100 μM stock and divided into aliquots. All inhibitors prepared in DMSO were stored at -20°C, then diluted in Ringer's solution just before use. A complete list of agents used can be found in Table 1.
Pharmacological agents used to uncover ions and molecular mediators involved in carbachol-induced pigment granule dispersion
L-type Ca2+ channel antagonist
10 μM, 100 μM
Intracellular Ca2+ chelator
1 μM, 30 μM
PP2B (calcineurin) inhibitor
100 nM, 0.5 μM, 1 μM
20 nM, 200 nM, 2 μM
Phorbol 12-myristate 13-acetate
1 μM, 10 μM, 100 μM
Drug concentrations used are justified by the articles referenced. All agents were purchased from Sigma-Aldrich (St. Louis, MO).
Microscopy and statistical analysis
Fixed RPE was placed onto a slide and chopped into smaller fragments using a coverslip. For each treatment sample, thirty cells were measured from each fish, and a minimum of three fish was used in each experiment. Measurements were made using a Zeiss phase-contrast light microscope equipped with an ocular micrometer. Cells were measured if they appeared to be whole, that is, if they had the phase-bright base typical of an intact cell and long apical processes giving an overall length of at least 50 μm. Cells were only measured if they had at least three apical processes.
A pigment index (PI) was calculated by dividing the distance from the base of the cell to the farthest dispersed pigment granule by the total length of the cell. Analysis of variance (ANOVA) was performed among the mean pigment indices of each treatment group. Differences were considered significant when p < 0.05. Tukey post hoc analysis was performed to determine which treatments differed. Prior to analysis, the two control conditions (FSK and carbachol-alone) were combined from all experiments. When experimental conditions were found statistically significantly different from the carbachol control condition, a percent inhibition was calculated. To calculate percent inhibition, first the difference between experimental treatment and FSK treatment was found. This value was then divided by the difference between FSK and carbachol conditions and multiplied by 100. Finally, the resulting value was subtracted from 100 to yield percent inhibition.
The authors are grateful for help with the statistical analysis provided by Dr. Butch Weckerly. The authors also wish to thank Dr. Joseph Koke for help in critically reading the manuscript. Finally, the authors thank Elizabeth Crittenden for support and training in the experimental techniques performed in this study and for the tissue used to generate Figure 1. This study was funded by research grants from the National Science Foundation (NSF IOB-0615762, IOB-0634704, IOB 05-33445 and IOB-0235523) and Hope for Vision.
Biology Department, Texas State University-San Marcos
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