Basic properties of beta adrenergic receptors mediating melanosome dispersion of melanophores in a cyprinid fish, Zacco temmincki

In melanophores of a cyprinid fish, Zacco temmincki, receptor mechanisms of melanosome dispersion induced by catecholamines were examined. While possessing a melanosome-aggregating action in higher concentrations, isoproterenol and epinephrine in lower concentrations acted to disperse melanosomes. Norepinephrine, epinine and dopamine altered their action from melanosome aggregation to melanosome dispersion after alpha adrenergic blockade. The catecholamine-induced melanosome dispersion was inhibited by beta adrenergic blocking agents. Mediation of dispersion is regulated through beta adrenergic receptors. The beta adrenergic responses were unaffected by mersalyl, a sulfhydryl inhibitor. A prospective substance acting in dispersing melanosomes appears to be adrenaline, but not noradrenaline.     

Regulation of Organelle Movement in Melanophores by Protein Kinase A (PKA), Protein Kinase C (PKC), and Protein Phosphatase 2A (PP2A)

We used melanophores, cells specialized for regulated organelle transport, to study signaling pathways involved in the regulation of transport. We transfected immortalized Xenopus melanophores with plasmids encoding epitope-tagged inhibitors of protein phosphatases and protein kinases or control plasmids encoding inactive analogues of these inhibitors. Expression of a recombinant inhibitor of protein kinase A (PKA) results in spontaneous pigment aggregation. α-Melanocyte-stimulating hormone (MSH), a stimulus which increases intracellular cAMP, cannot disperse pigment in these cells. However, melanosomes in these cells can be partially dispersed by PMA, an activator of protein kinase C (PKC). When a recombinant inhibitor of PKC is expressed in melanophores, PMA-induced pigment dispersion is inhibited, but not dispersion induced by MSH. We conclude that PKA and PKC activate two different pathways for melanosome dispersion. When melanophores express the small t antigen of SV-40 virus, a specific inhibitor of protein phosphatase 2A (PP2A), aggregation is completely prevented. Conversely, overexpression of PP2A inhibits pigment dispersion by MSH. Inhibitors of protein phosphatase 1 and protein phosphatase 2B (PP2B) do not affect pigment movement. Therefore, melanosome aggregation is mediated by PP2A.     

The stimulatory effects of prostaglandins on the melanophores of the lizard, Anolis carolinensis

The melanosome dispersing activity of prostaglandins PGE1, PGE2, PGF1 alpha, PGF2 alpha, PGI2 and 6 beta PGI, was tested on the melanophores of Anolis carolinensis. Only PGE2 and PGE1 were active and while PGE2 was the most potent and acted synergistically with alpha-MSH, PGE1 was additive with alpha-MSH. Arachidonic acid also stimulated melanosome dispersion but its effect was blocked by indomethacin suggesting an action through its conversion to PGE1 or PGE2. The effect of alpha-MSH, on the other hand, was unaltered by indomethacin which suggests that alpha-MSH stimulated melanosome dispersion does not depend upon prostaglandin synthesis. Thus, while some prostaglandins may interact with alpha-MSH to stimulate melanosome dispersion they are unlikely to mediate its action.     

Melanophores of Zacco temmincki (Teleostei) are light sensitive

The responses of melanophores of a cyprinid fish Zacco temmincki to changes in illumination were examined in isolated scale preparations of the adult fishes. Melanosomes in the melanophores aggregated in darkness and dispersed in light. These responses were invariably induced, even in denervated melanophores. These light responses, the dark-induced aggregation and the light-induced dispersion, were not affected by a number of alpha and beta adrenergic blocking agents. It was concluded that the melanophores of Zacco temmincki were themselves light sensitive and responded directly to light by melanosome translocations. The light responses were quantitatively assessed in relation to the intensity of illumination.     

Physiological Color Changes in Reptiles

SYNOPSIS. The physiological regulation of color changes in reptilesas studied in the lizard, Anolis carolinensis, is discussed.In Anolis, the ability to adapt to a background is dependentupon the level of circulating MSH, therelease of which is dependenton information received through the eyes. Blinded (or intact)lizards are brown under conditions of strong illumination andgreen under conditions of lower light intensities, and, again,these color changes are regulated by MSH. According to Kleinholz,color changes in the blinded lizard are regulated by dermalphotoreceptors. High or low temperatures directly affect thecolor of Anolis skins and alter the rate at which skins respondto hormones. Aggregationof melanin granules within Anolis melanophoresin response to sympathomimetic stimulation is regulated throughalpha adrenergic receptors whereas dispersion of melanin granulesin response to such stimulation is controlled through beta adrenergicreceptorspossessed by the melanophores. Most Anolis melanophores possessboth alpha and beta adrenergic receptors, but some melanophorespossess only beta adrenergic receptors. In the normal physiologyof the lizard, under conditions of stress, stimulation of alphaadrenergic receptors by catecholamines leads to an "excitement—pallor"followedby an "excitement—darkening" resulting from stimulationof beta adrenergic receptors which causes dispersion of melaningranules within localized populations of melanophores. Thus,in Anolis, dispersion of melanin granules within melanophoresis regulated by both MSH and by catecholamines. Evidence ispresented that the intracellular level of cyclic 3', 5'-AMPwithin melanophores may be responsible for the regulation ofmovement of melanin granules.     

A rapid quantitative bioassay for evaluating the effects of ligands upon receptors that modulate cAMP levels in a melanophore cell line.

A new method for rapidly evaluating the effects of drugs on receptors that regulate intracellular cAMP in a cell line derived from Xenopus laevis melanophores has been developed. Melanophores were plated into sterile 96 well microtiter plates, and 3 days later the cells were treated with melatonin for 30 min to induce melanosome aggregation. Subsequent exposure to MSH or adrenergic agonists caused dose dependent pigment dispersion that peaked within 30 min. The cumulative pigment displacement from cells could be quantitated by using a microplate reader to measure changes in transmittance of light through the wells. The acquired data enabled detailed and reproducible dose response curves and time course analyses to be generated. In addition, the assay followed for the rapid characterization of the effects of antagonists upon the beta adrenergic receptor (beta AR). The assay has the potential to test the effects of ligands upon any receptor capable of mediating pigment translocation in the melanophore cell line.     

The protein-phosphatase inhibitor okadaic acid mimics MSH-induced and melatonin-reversible melanosome dispersion in Xenopus laevis melanophores.

The present study describes the ability of 315 nM okadaic acid to induce melanosome dispersion in cultured Xenopus laevis melanophores. This effect of okadaic acid is similar to that of a-melanocyte stimulating hormone (MSH) and can be reversed by melatonin treatment; it indicates that a member of the protein-phosphatase 1 or 2A families must be active for maintenance of the aggregated state. Higher concentrations of okadaic acid (1 microM) attenuate the response of Xenopus melanophores to melatonin leading to the hypothesis that melatonin action is mediated by the calcium/calmodulin activated phosphatase 2B. This hypothesis seems unlikely, however, since the calcium/calmodulin inhibitors TFP and W7 do not prevent melatonin-induced pigment aggregation, but instead induce aggregation on their own.     

Receptor mechanisms in fish chromatophores--VIII. Mediated by beta adrenoceptors, catecholamines always act to disperse pigment in siluroid melanophores

The sympathomimetic amines tested, including those of alpha and beta type, were all ineffective in arousing melanosome aggregation within dermal and epidermal melanophores of the siluroid catfish, Parasilurus asotus. Conversely, these amines unfailingly gave rise to a dispersion of the pigment. While alpha-adrenergic blocking agents had only a little influence, beta agents exhibited a strong inhibitory effect on the pigment-dispersing action of the amines. Electrical nervous stimulation failed to bring about a melanosome dispersion. It was concluded that the adrenoceptors possessed by dermal and epidermal melanophores of this species seemed to be solely of the beta-adrenergic type, mediating the pigment dispersion, and that the endogenous amines involved in the darkening reaction of animals may originate in adrenal chromaffin cells, and thus are not derived from the peripheral nervous elements.     

The Protein-Phosphatase Inhibitor Okadaic Acid Mimics MSH-Induced and Melatonin-Reversible Melanosome Dispersion in Xenopus laevis Melanophores

The present study describes the ability of 315 nM okadaic acid to induce melanosome dispersion in cultured Xenopus laevis melanophores. This effect of okadaic acid is similar to that of a-melanocyte stimulating hormone (MSH) and can be reversed by melatonin treatment; it indicates that a member of the protein-phosphatase 1 or 2A families must be active for maintenance of the aggregated state. Higher concentrations of okadaic acid (1 μM) attenuate the response of Xenopus melanophores to melatonin leading to the hypothesis that melatonin action is mediated by the calcium/calmodulin activated phosphatase 2B. This hypothesis seems unlikely, however, since the calcium/calmodulin inhibitors TFP and W7 do not prevent melatonin-induced pigment aggregation, but instead induce aggregation on their own.     

Rab32 regulates melanosome transport in Xenopus melanophores by protein kinase a recruitment

Intracellular transport is essential for cytoplasm organization, but mechanisms regulating transport are mostly unknown. In Xenopus melanophores, melanosome transport is regulated by cAMP-dependent protein kinase A (PKA). Melanosome aggregation is triggered by melatonin, whereas dispersion is induced by melanocyte-stimulating hormone (MSH). The action of hormones is mediated by cAMP: High cAMP in MSH-treated cells stimulates PKA, whereas low cAMP in melatonin-treated cells inhibits it. PKA activity is typically restricted to specific cell compartments by A-kinase anchoring proteins (AKAPs). Recently, Rab32 has been implicated in protein trafficking to melanosomes and shown to function as an AKAP on mitochondria. Here, we tested the hypothesis that Rab32 is involved in regulation of melanosome transport by PKA. We demonstrated that Rab32 is localized to the surface of melanosomes in a GTP-dependent manner and binds to the regulatory subunit RIIalpha of PKA. Both RIIalpha and Cbeta subunits of PKA are required for transport regulation and are recruited to melanosomes by Rab32. Overexpression of wild-type Rab32, but not mutants unable to bind PKA or melanosomes, inhibits melanosome aggregation by melatonin. Therefore, in melanophores, Rab32 is a melanosome-specific AKAP that is essential for regulation of melanosome transport.     

Spectral sensitivity of melanophores of a freshwater teleost, Zacco temmincki

1. The melanophores of a freshwater teleost, Zacco temmincki, responded to changes in illumination: in darkness the melanophores induced a melanosome aggregation and when subjected to light they caused a melanosome dispersion. 2. Using monochromatic light, the spectral sensitivity of the melanophores was examined. 3. The melanophores showed a different sensitivity to light between 400 and 600 nm with a maximum at about 525 nm. 4. The action spectrum closely resembled a porphyropsin absorbance curve, suggesting a porphyropsin or similar photopigment is active in the melanophore light response of Zacco temmincki.     

A Rapid Quantitative Bioassay for Evaluating the Effects of Ligands Upon Receptors That Modulate cAMP Levels in a Melanophore Cell Line

A new method for rapidly evaluating the effects of drugs on receptors that regulate intracellular cAMP in a cell line derived from Xenopus laevis melanophores has been developed. Melanophores were plated into sterile 96 well microtiter plates, and 3 days later the cells were treated with melatonin for 30 min to induce melanosome aggregation. Subsequent exposure to MSH or adrenergic agonists caused dose dependent pigment dispersion that peaked within 30 min. The cumulative pigment displacement from cells could be quantitated by using a microplate reader to measure changes in transmittance of light through the wells. The acquired data enabled detailed and reproducible dose response curves and time course analyses to be generated. In addition, the assay followed for the rapid characterization of the effects of antagonists upon the (β adrenergic receptor (β AR). The assay has the potential to test the effects of ligands upon any receptor capable of mediating pigment translocation in the melanophore cell line.     

Effect of colcemid on the centrosome and microtubules in dermal melanophores of Xenopus laevis larvae in vivo.

An electron microscopy study showed that in melanophores with dispersed and aggregated pigment the sensitivity of the centrosome and the stability of microtubules were different and depended on the colcemid concentration. The structure of the centrosome didn't change upon exposure to colcemid in dispersed melanophores. In aggregated melanophores, on exposure to 10(-6) M colcemid, the centrosome retained its structure; colcemid at 10(-5)-10(-3) M caused a dramatic collapse of the centrosome. Treatment of aggregated melanophores with colcemid resulted in the complete disassembly of the microtubules; though microtubules in dispersed melanophores appear to be colcemid resistant. Light microscopy studies indicated that in Xenopus melanophores with aggregated or dispersed pigment melanosomes didn't change their location after exposure to 10(-3)-10(-6) M colcemid. Subsequent incubation in colcemid-free medium revealed that the cells retained their ability to translocate melanosomes in response to hormone stimulation. Electron microscopy data revealed the inactivation of the centrosome as MTOC (microtubule-organizing center) in dispersed melanophores with melatonin substituted for MSH in the presence of colcemid. In contrast, with melanocyte-stimulating hormone (MSH) substituted for melatonin, we observed the activation of the centrosome in aggregated cells. We showed that in aggregated melanophores pigment movement proceeded in the complete absence of microtubules, suggesting the involvement of a microtubule-independent component in the hormone-induced melanosome dispersion. However, we observed abnormal aggregation along colcemid-resistent microtubules in dispersed melanophores, suggesting the involvement of not only stable but also labile microtubules in the centripetal movement of melanosomes. The results raise the intriguing questions about the mechanism of the hormone and colcemid action on the centrosome structure and microtubule network in melanophores with dispersed and aggregated pigment.     

Spectral Sensitivity of Melanophores in the Primary Color Response of the Rose Bitterling, <Emphasis Type="Italic">Rhodeus ocellatus ocellatus</Emphasis>

Melanophores of young Rhodeus ocellatus ocellatus have the ability to respond by melanosome dispersion to the direct action of visible light. The effective wavelength within visible light region for inducing melanosome dispersion was investigated using melanophores located around the base of the dorsal fin of young fish set on the stage of a light microscope. The melano- -’ phores were exposed to light of various wavelengths (420–680 nm) but of the same intensity by placing interference filters under the condenser diaphragm. The most effective wavelength was about 420 nm. Longer wavelengths were less effective for the induction of melanosome dispersion.     

The role of calcium in MSH stimulated melanosome dispersion

We have examined the role of the calcium ion in the response of the melanophores of the lizard, Anolis carolinensis to alpha-melanocyte stimulating hormone (alpha-MSH) by a rate method which allows kinetic analysis of melanosome dispersion. Dose-response curves to alpha-MSH were compared in the presence of different calcium concentrations, and Schild regression plots were constructed. An avidly binding analogue of alpha-MSH, Nle4-D-Phe7-alpha-MSH, was used to demonstrate kinetically the involvement of calcium in the melanophore response both for MSH receptor binding and the subsequent transduction of the MSH signal across the melanophore membrane.     

Melanin concentrating hormone (MCH) effects on teleost (Chrysiptera cyanea) melanophores

The in vitro biological actions of synthetic chum salmon melanin concentrating hormone (MCH) on melanophores of the blue damselfish (a teleost), Chrysiptera cyanea, were studied. This cyclic heptadecapeptide stimulated melanosome (melanin granule) aggregation (centripetal migration) within melanophores at a threshold concentration of about 10(-10) M. The action of this putative hormone was not blocked by alpha- or beta-adrenoceptor antagonists. It was concluded that the effects of MCH were direct and were not mediated indirectly through the actions of adrenergic neurotransmitters released from nerve terminals. Further evidence for this view comes from the observation that, unlike the case of neurotransmitter release, melanosome aggregation in response to MCH proceeded in the absence of calcium. The possible role of MCH in the control of color change of teleost fishes is discussed.     

An increase in extracellular Ca(2+) concentration induces pigment aggregation in teleostean melanophores

An increase in the concentration of Ca(2+) ions in the external medium ([Ca(2+)](o)) induced pigment aggregation in melanophores of three species of freshwater teleosts examined. Denervated melanophores were refractory to elevations of [Ca(2+)](o). The pigment-aggregating action was inhibited by the sympathetic blocking agents, phentolamine, prazosin and yohimbine. Bretylium, an agent known to block the release of the neurotransmitter, interfered with the response effectively. Ca(2+) blockers, such as Mn(2+), verapamil and gallopamil, also inhibited the response, possibly by inhibiting Ca(2+) entry into the presynaptic elements of melanosome-aggregating fibers. The conclusion is that the increase in [Ca(2+)](o) may induce membrane depolarization of presynaptic nervous elements around the melanophores, which open the voltage-dependent Ca(2+) channels there. The liberation of adrenergic neurotransmitter follows, which induces the aggregation of pigment in melanophores.     

TWO ACTIONS OF CYCLIC AMP ON MELANOSOME MOVEMENT IN FROG SKIN : Dissection by Cytochalasin B

Photomicrography and reflectance microphotometry were used to monitor melanosome movement in frog skin melanocytes in vitro in response to hormonal stimulation and cytochalasin B (CB). Melanocyte-stimulating hormone (MSH), theophylline, and dibutyryl cyclic AMP (DiBcAMP) induced melanosome dispersion (darkening) which was promptly arrested by cytochalasin B in concentrations of 5–20 µg/ml. Melanosome aggregation (skin lightening) occurred only after removal of the darkening agent (MSH, theophylline, or DiBcAMP) and proceeded in the presence or absence of CB. When CB was added to darkened skins, they did not lighten and melanosomes remained in the dispersed state. Use of CB has permitted the dissection of cyclic AMP-mediated melanosome dispersion into two distinct events. The first, induction of melanosome dispersion, is CB sensitive. The second action of intracellular cyclic AMP involves an uncoupling of the centripetal motive force, and is CB insensitive. In the latter process, production of cyclic AMP appears to produce the same result as application of microtubule-disrupting agents.     

Subtypes of beta adrenergic receptors mediating pigment dispersion in chromatophores of the medaka, Oryzias latipes

Actions of the adrenergic beta-2 agonists, salbutamol and terbutaline, and the beta-1 antagonists, metoprolol and atenolol, were examined on denervated melanophores and leucophores of a teleost, Oryzias latipes. Beta-2 agonists depressed the pigment-aggregation response of melanophores to norepinephrine, while beta-1 antagonists inhibited the dispersion response of leucophores to isoproterenol but not the melanophore response. These findings suggest that adrenergic receptors mediating pigment dispersion in melanophores are beta-2 and those of leucophores are beta-1. The possible relations between receptor mechanisms and the responses of chromatophores are discussed.     

Cellular Aspects of the Control of Physiological Color Changes in Amphibians

SYNOPSIS. The present paper is concerned mainly with the melanin-dispersingeffect of melanocyte-stimulating hormones (MSH's) on the skinmelanophores of amphibians. In addition, some of the more recentevidence for the unihumoral theory of the control of color changeis reviewed. The mechanism of dispersion of melanin is stillunknown, but evidence is accumulating that the action of MSHmay be mediated by an increase in the melanophoric content ofadenosine 3', 5'-monophosphate (cyclic AMP). For example, cyclicAMP has a specific, reversible melanin-dispersing effect onthe melanophores of the isolated skin of R. pipiens and Xenopuslaevis. It also has a reversible "melanophore—expanding"effect on the tissue—cultured embryonic melanophores ofthe spotted salamander, Ambystoma maculatum. The effect of cyclicAMP on melanophores of R. pipiens does not require sodium butis inhibited by hypertonicity. Finally, new evidence is presented that confirms that the melanin-dispersingeffect of catecholamines on melanophores of X. laevis is mediatedby beta adrenergic receptors,because it is blocked by the highlyspecific ß—blocking agent, propranolol. On theother hand, the melanin-aggregating effect of catecholamineson amphibian melanophores appears to be mediated by alpha adrenergicreceptors. There is even a possibility that the effects of catecholaminesare also mediated through a control of cyclic AMP levels inmelanophores, with beta adrenergic stimulation producing anincrease in cyclic AMP levels, followed by dispersion of melanin,and alpha adrenergic stimulation producing a decrease in cyclicAMP levels, followed by aggregation of melanin.