Here we show that carbachol induces pigment granule dispersion in RPE isolated from bluegill (Lepomis macrochirus). The maximal effect of carbachol was seen at concentrations as low as 10 nM. This result is consistent with García's [9] earlier study in which carbachol induced pigment granule dispersion in isolated RPE of green sunfish (Lepomis cyanellus). Using bluegill, we have shown that carbachol-induced pigment granule dispersion is not species-specific and that carbachol-induced pigment dispersion is dose-dependent and saturable, indicating a receptor-mediated process. Atropine, a general muscarinic antagonist, was shown to block pigment granule dispersion, suggesting muscarinic receptors mediate carbachol-induced dispersion. Furthermore, because M1 and M3 antagonists strongly inhibited pigment granule dispersion induced by carbachol and because the M1 agonist 4-CP induced pigment granule dispersion, the involvement of M1 or M3 receptors is suggested. The involvement of an M5 receptor cannot be addressed pharmacologically at this time since no M5-selective agents are currently commercially available.
Although the results from the pharmacological studies with muscarinic receptor agonists and antagonists are internally consistent with the interpretation that Modd receptors are involved in mediating carbachol-induced dispersion (Modd reagents are effective; Meven are not), drug studies in embryonic chick have illustrated that pharmacological results taken by themselves can be misleading. Pirenzepine, although described as an M1-selective antagonist in both mammals and trout [15, 16], avidly binds M2 receptors in chick [16] and may bind M4 receptors in other systems as well [17]. Furthermore, Hsieh and Liao [11] have reported that pirenzepine binds the M2 receptor they have isolated from zebrafish with a submicromolar dissociation constant. Therefore, a molecular characterization of the receptor subtype(s) expressed by bluegill RPE must be done before a definitive assignment can be made. These studies are currently underway in our laboratory.
Given the ambiguity associated with the pharmacology of muscarinic receptors in non-mammalian vertebrates, the question of which muscarinic receptor is involved in carbachol-induced pigment granule dispersion remains open. Based on our current knowledge of the signaling pathways involved in regulating pigment granule movement, the simplest model for muscarinic receptor involvement would invoke an Meven receptor negatively coupled to adenylyl cyclase through Ginhibitory proteins (Figure 4). According to this model, activation of the receptor would lead to inhibition of adenylyl cyclase, resulting in decreased cAMPi levels due to degradation by phosphodiesterases, efflux via organic anion transporters, or both. As a consequence of decreased cAMPi, the activity of cAMP-dependent protein kinase (PKA) would diminish. The activity of protein phosphatases would then tip the balance between phosphoproteins and their dephosphorylated counterparts toward the latter, and this, in turn, would lead to pigment granule dispersion.
Consistent with this model are the observations that treatments expected to elevate cAMPi induce pigment granule aggregation in RPE isolated from bluegill (Table 1), green sunfish [2, 14, 18], and blue-striped grunt [19]. Furthermore, King-Smith et al. [20] demonstrated that simply washing away exogenously applied, extracellular cAMP was sufficient to induce pigment granule dispersion in both isolated RPE sheets and dissociated RPE cells. Importantly, elevation of cAMP caused aggregation and washout of cAMP caused dispersion irrespective of the external or internal concentrations of calcium. We have also observed exogenous cAMP induces aggregation in RPE isolated from bluegill, and its washout is sufficient to induce dispersion (García, unpublished observations). Presumably, in both bluegill and green sunfish RPE removing extracellular cAMP not only eliminated the supply of cAMP available for import via organic anion transporters [18], but also reversed the gradient, favoring its export. In addition, microinjection of the PKA inhibitor PKI5–24 amide into dissociated cells isolated from green sunfish resulted in pigment granule dispersion (García, unpublished observations).
The central importance of cAMP in regulating pigment granule position is also suggested by studies conducted on dermal melanophores of tilapia (Tilapia mossambica) [21–25], black tetra (Gymnocorymbus ternetzii) [26], and angelfish (Pterophyllum scalare) [27] among others (reviewed in [28]). In each of these cases, increased cAMPi is associated with pigment granule dispersion and decreased cAMPi with pigment granule aggregation. Studies done on permeabilized melanophores from tilapia demonstrated that the direction of pigment granule movement was dictated by the addition and removal of cAMP [22] and that pigment granule dispersion could be induced by addition of the catalytic subunit of PKA [25]. Similarly, in melanophores isolated from black tetra cAMP levels measured immunohistochemically tracked closely with extent of pigment granule movement toward the plus-end of microtubules during pigment granule dispersion [26]. Sammak et al. [27] observed fluxes in cAMPi as well as Ca2+ levels in melanophores isolated from angelfish, but found that only the former were necessary and sufficient to influence pigment granule position, whereas the latter were neither necessary nor sufficient to influence pigment granule movement. Similarly, Kotz and McNiven [29] presented pharmacological evidence suggesting that reducing cAMPi was sufficient to stimulate aggregation in squirrelfish melanophores, while Ca2+ dynamics appeared variable and unimportant.
Although the simplest model for muscarinic regulation would be through G-protein-mediated inhibition of adenylyl cyclase, a model involving activation of phospholipase C must also be considered. While evidence exists in heterologous expression systems for M1 receptor-based inhibition of adenylyl cyclase [17], Modd receptor activation is more typically associated with activation of phospholipase C. In general, phospholipase C catalyzes the conversion of phosphatidylinositol bisphosphate to diacylglycerol and inositol trisphosphate (IP3). IP3, in turn, activates ligand-gated Ca2+ channels located on intracellular storage organelles, leading to release of Ca2+ from these stores, and an elevation of cytoplasmic free Ca2+. Cytoplasmic Ca2+ typically binds to and activates calmodulin, which modulates the activities of a number of enzymes, including certain phosphodiesterases (e.g. PDE1) as well as some protein phosphatases (e.g. calcineurin). In the former case, increasing phosphodiesterase activity would decrease cAMP levels, leading to decreased PKA activity and decrease in the rate of phosphorylation of PKA-target proteins. In addition, if protein phosphatases were activated, the tendency for target proteins to be dephosphorylated would be further enhanced. It should be noted that calcineurin has been shown to be involved in regulating pigment granule movement in melanophores isolated from tilapia [25]. In either case (or both cases), the balance would be shifted to dephosphorylated proteins which, though not yet identified, seem to favor pigment granule dispersion.
The results we present herein are consistent with this model in that agents characterized as Modd-selective in other systems affected the pigment granule movement under study, while Meven-selective agents did not [10]. However, one wonders how to reconcile these results with those reported for green sunfish RPE [20] or melanophores [27, 29] showing that Ca2+ transients are neither necessary nor sufficient for pigment granule movement in either direction, i.e. aggregation or dispersion. One possibility is that the Ca2+ "requirement" for pigment granule dispersion in RPE can be bypassed or overruled by other mechanisms. For example, King-Smith et al. [20] observed that in RPE isolated from green sunfish pigment granule dispersion initiated by cAMP-washout could be accomplished in the absence of extracellular Ca2+, following depletion of intracellular stores by incubating RPE sheets in Ca2+-free medium, or damping changes in intracellular Ca2+ by infiltrating the cells with the Ca2+-chelator BAPTA. In any of these cases, cAMPi may have been attenuated in the cells by efflux through the activity of organic anion transporters in addition to basal activity of phosphodiesterases. Thus, a requirement for Ca2+-based reduction in either cAMP or phosphoproteins could be bypassed by a mechanism involving cAMP-efflux. RPE isolated from green sunfish seem to have a high basal phosphatase activity since treatment with okadaic acid is sufficient to induce pigment granule aggregation (García, unpublished results).
King-Smith et al. [20] also showed that elevating Ca2+ by treating cells with ionomycin was not sufficient to prevent aggregation when 1 mM cAMP was added to the medium. Earlier work done by García and Burnside [18] suggested that cAMP was imported into RPE cells via organic anion transporters. Thus, given a sufficient gradient for import, cAMP levels in the cell could remain high along with PKA activity, resulting in a continual phosphorylation of target proteins associated with pigment granule aggregation, even in the face of increased phosphodiesterase activity or phosphatase activity.
Also consistent with the results we report here suggesting that bluegill RPE express muscarinic receptors are the observations that human [30, 31] and rat [32] RPE express muscarinic receptors. The receptors on human RPE have been linked to phosphoinositide hydrolysis [30, 31], suggesting that they belong to the M1, M3 or M5 subclass or some combination of these subclasses. Based on pharmacological studies, Feldman et al. [30] concluded that M3 receptors are the subtype present on human RPE.