Pigment Dispersion in Frog Melanophores Can Be Induced by a Phorbol Ester or Stimulation of a Recombinant Receptor That Activates Phospholipase C*

Pigment dispersion in frog melanophores is classi- cally mediated by receptors that activate protein kinase A via an elevation of intracellular cyclic AMP. Here, 12-0-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C, is found to induce pigment dispersion. To demonstrate that an increase in cAMP is not required for the melanosome movement, a murine bombesin receptor was expressed in the melanophores. When these cells were treated with bom- besin, they accumulated intracellular inositol phosphates but not cAMP and dispersed their pigment. Four agonists, one partial agonist, and two antagonists for the bombesin receptor were compared for their ability to induce or block bombesin-induced pigment dispersion, In all cases, the degree of pigment dispersion followed simple equilibrium reactions. The resulting dose-response curves allowed for the determination of the effective concentration for half-maximal pigment dispersion (ECBo) or half-maximal inhibition of bom- besin-stimulated pigment dispersion (ICeo) for the peptides. As the pigment dispersion assay can rapidly eval- uate chemicals for their effects on receptors that activate phospholipase C via a functional assay, it has potential utility for investigations of ligand-receptor the peak labeled as IP3 coeluted with [3H]1,4,5-IP3. The lack of more detailed study precluded absolute identification of the peaks because of the potential for coelution of other untested isomers.


Pigment dispersion in frog melanophores is classically mediated by receptors that activate protein kinase A via an elevation of intracellular cyclic AMP.
Here, 12-0-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C, is found to induce pigment dispersion. To demonstrate that an increase in cAMP is not required for the melanosome movement, a murine bombesin receptor was expressed in the melanophores. When these cells were treated with bombesin, they accumulated intracellular inositol phosphates but not cAMP and dispersed their pigment. Four agonists, one partial agonist, and two antagonists for the bombesin receptor were compared for their ability to induce or block bombesin-induced pigment dispersion, In all cases, the degree of pigment dispersion followed simple equilibrium reactions. The resulting dose-response curves allowed for the determination of the effective concentration for half-maximal pigment dispersion (ECBo) or half-maximal inhibition of bombesin-stimulated pigment dispersion (ICeo) for the peptides. As the pigment dispersion assay can rapidly evaluate chemicals for their effects on receptors that activate phospholipase C via a functional assay, it has potential utility for investigations of ligand-receptor interactions and for massive drug screening.
Many vertebrates possess the ability to rapidly change their skin color. Depending on the animal, a variety of chromatophores, including melanophores, xanthophores, erythrophores, and iridophores, are involved in this process. Color changes can be mediated by a variety of stimuli including direct photostimulation, hormonal regulation, and neuronal activity. The best characterized pathway for controlling pigment movement utilizes the cAMP second messenger system, and it has been rigorously demonstrated that an elevation above the base-line in intracellular cAMP is sufficient to cause pigment dispersion. Increasing cAMP is associated with phosphorylation of a 57-kDa protein and centrifugal pigment translocation while a dimunition in cAMP leads to dephosphorylation of the protein and centripetal melanosome movement (1, 2). In addition to the role of cAMP in the control of pigment movement, a few recent studies have examined the potential roles of inositol 1,4,5-trisphosphate (1,4,5-IP3)' and diacylglycerol (DAG) in this process. In one investigation, 1,4,5-IP3 was found to induce pigment aggregation in melanophores in a species of fish (3). In a second report, the mechanism by which melanin concentrating hormone works in an eel was described (4). In this latter report, 1-10 nM 12-0-tetradecanoylphorbol-13-acetate (TPA) induced skin lightening while higher concentrations of up to 10 p~ had no effect on coloration.
One of the most popular types of cells for investigating the basis of pigment translocation, as well as chemicals which stimulate melanosome dispersion or aggregation, are melanophores from frogs. From studies with both frog skin and immortalized pigment cells, it is known that an increase in intracellular cAMP leads to pigment dispersion over the course of several minutes. Some examples of agents known both to raise cAMP levels in the frog cells and cause pigment dispersion include a-melanocyte-stimulating hormone (a-MSH), P-adrenergic agents, light, and forskolin (5)(6)(7)(8)(9). Melatonin, on the other hand, decreases intracellular cAMP levels and leads to pigment aggregation (6,lO). However, pathways that do not involve modulation of cAMP levels, such as that involving phospholipase C and the generation of inositol phosphates, have not been examined for their potential role in effecting pigment movement. Here we show that pigment dispersion can be induced by TPA, a potent activator of protein kinase C. In addition, we show that activation of a recombinant murine bombesin receptor expressed in melanophores leads to phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis and pigment dispersion in the absence of an elevation in intracellular CAMP. Cell Culture-The propagation of Xenopus lueuis melanophores and fibroblasts was performed as described earlier (9,11). The melanophores used in this paper were derived from a clonal cell line isolated from a primary culture.
Plasmid DNA Constructs-The expression vector pJG3.6 was constructed from pBluescript I1 SK(-) (Bluescript, Stratagene) and pcDNAI/Neo (Invitrogen) as follows. A 2.0-kb plasmid fragment in Bluescript containing the ampicillin resistance gene and the ColEl origin of replication was amplified by PCR, using Vent DNA polymerase (New England Biolabs, Inc.), the sense primer from nucleotides 960 to 974 (5'-GACGTCGACGGAAACCTGTCGTGC-3') and the antisense primer from nucleotides 1 to 15 (5"GATATCGA-TAGGGCGCGTCAGGTG-3'). The restriction enzyme sites AatII and SalI were engineered into the 5' end of the former primer and EcoRV and ChI into the latter to aid with subsequent ligations. A 1.7-kb plasmid fragment encompassing the cytomegalovirus promotor, multicloning site, and poly(A) region in pcDNAI/Neo was amplified by PCR methodology as described above using the sense primer from nucleotides 1525 to 1539 (5"GATATCGATGGCCAGATA-TACGCG-3') and the antisense primer from nucleotides 3179 to 3193 (5'-ACGTCGACGGGATCGGGAGATCC-3'). Again, the restriction enzyme sites EcoRV and ChI were engineered into the 5' end of the former primer and SalI and Aut11 into the latter. The PCR products were size-fractionated on an agarose gel, and the appropriate bands were cut from the gel and purified following the Geneclean II Kit protocols (Bio 101 Inc.). Both fragments were digested with SalI and ChI and ligated together to form the expression vector pJG3.6. The ligation mixture was transformed into electrocompetent bacteria DH5a (Gibco Bethesda Research Laboratories) by electroporation (12,13). The SBR expression vector (pcDNAI/NeoBR), prepared by cloning the HindIII-digested SBR gene into the HindIII site of the expression vector pcDNAI/Neo, was kindly donated by Jim Battey (14). The SBR gene was subcloned into pJG3.6 (pJG3.6BR) by digesting pc-DNAI/NeoBR and pJG3.6 with HindIII, band purifying the appropriate fragments, and then ligating them together. The vector was then transformed into DHlOB bacteria (Gibco Bethesda Research Laboratories) by electroporation. The expression vector pON260, which contains the P-galactosidase (lacZ) gene under the transcriptional regulation of a cytomegalovirus promoter, was generously supplied by Susan Amara (15). cDNA coding for the TRH receptor was a generous gift from Marvin Gershengorn (16). It was subcloned into pJG3.6 by ligation of the 3.5-kb ApaI/BamHI-digested TRH receptor gene fragment into ApaIIBarnHI-digested pJG3.6.
The substance P receptor gene was cloned into pJG3.6 (pJG3.6SubP) as follows. The substance P receptor cDNA was isolated from a rat olfactory epithelieum cDNA library using PCR, Taq DNA polymerase (Perkin-Elmer Cetus), and oligonucleotide primers based on the cDNA sequence reported for the rat substance P receptor (17). The sense primer spanned the nucleotide region from -549 to -532 (5'-ACCAAGACGGACAAGCTG-3') and the antisense primer spanned the nucleotide region from 1446 to 1463 (5"GCAAGGA-TAGCCTTCTCA-3'). A 2-kb pCR product was visible after size fractionation on agarose gel. This fragment was band purified using Geneclean and ligated into the pCR 1000 vector (Invitrogen) following the TA Cloning System protocols (Invitrogen). The ligation mixture was transformed into INVaF' cells following the TA Cloning System protocols (Invitrogen), plated on LB agar dishes, and grown overnight at 37 "C. A total of 19 white colonies were selected for screening with PCR using pUC/M13 reverse and forward primers. Of the 19 colonies, three gave PCR fragments of approximately 2 kb. Restriction enzyme digest analysis of the PCRproducts confirmed that all three contained the substance P receptor gene. The 2-kb cDNA fragment encoding the substance P receptor was excised from pCRlOOO by digestion with HindIII and HincII, isolated after size fractionation by band excision, and purified using Geneclean. The fragment was directionally ligated into HindIII/EcoRV-digested pcDNAI/Neo that had been previously treated with bacterial alkaline phosphatase and band purified as described above. The ligation mixture was transformed into the bacteria MC1061/P3 (Invitrogen) by electroporation. The substance P receptor gene was subcloned into pJG3.6 by digesting both plasmids with the restriction enzymes HindIII and XbaI and purifying the appropriate fragments. The fragments were ligated together and transformed into the bacteria DHlOB by electroporation. Plasmid constructs were verified by restriction enzyme digest analysis and plasmid DNA was isolated and purified using standard protocols (18).
Transfection-Transient expression of plasmid DNA in melanophores was achieved via electroporation (12, 13) using a Gene Pulsar apparatus (Bio-Rad) at a capacitance setting of 960 microfarad and voltage setting of 0.4 kV. The following procedures were performed on ice or at 4 "C. Cells were washed with 70% PBS, detached by applying 2.5% trypsin (Gibco) containing 3 mM EDTA (Baker) in 70% PBS, and quenched with 70% L-15 (Sigma) containing 20% (v/ v) calf frog serum (Gibco). The cells were collected by centrifugation for 5 min at 2000 X g, the medium was removed followed by resuspension in 10 ml of 70% PBS, and the total number of cells determined using a hemocytometer. The cells were then centrifuged as above, the supernatant removed, the cells were resuspended in 70% PBS (pH 7.0) at a concentration of 2.5 X lo6 cells/ml and placed in a pre-chilled electroporation cuvette (0.4-cm gap, Bio-Rad). The appropriate expression vector plasmid (40 pg) was then added to the cuvette in a volume of 10 pl. The cells were triturated 5 or 10 min after addition of the vector and once again immediately before electroporation (10 or 20 min after vector addition). Immediately after electroporation, the cells were transferred to fibroblast-conditioned growth medium (1.5-12 ml) which consisted of 70% L-15 supplemented with 20% fetal calf serum (Gibco). Cell death ranged from 40 to 60%, and the range of transient transfection efficiency was 10-60% but averaged about 30%. SBR transfection efficiency was generally assessed 3 days after electroporation by observing the number of cells that dispersed their pigment in response to 100 nM bombesin. To ensure that transfection efficiency was homogenous when preparing identical experiments, electroporations were pooled together before plating.
Microtiter Plate Assays-After electroporation, cells were seeded into either flat bottom 96-well tissue culture plates (Falcon) or GCSS 96-well tissue culture plates (SLT Lab Instruments). The plates were seeded either at or slightly below confluency (5,000-7,000 cells/GCSS well or 12,000-15,000 cells/flat bottom well) using a volume of 100 pl/well. After the cells had attached to the well surface (1-2 h), the medium was removed by aspiration and replaced with 100 pl of conditioned growth medium. In some instances, this procedure was performed the next day. The cells were incubated at 27 "C for 2 days following electroporation. Except as noted, the medium was removed by aspiration after 48 h and the cells washed with 100 pl of 70% PBS or 70% EX-CELL 320 part A containing 1% part B and no sodium bicarbonate (EX-CELL; JRH Biosciences), a defined serum-free medium. The cells were then incubated overnight in 100 p1 of EX-CELL per well. Wild type melanophores used for the controls were plated at or slightly below confluency (5,000-7,000 cells/GCSS well or 12,000-15,000 cells/standard well) 2-3 days before assaying. A day before the assay, the cells were washed with and incubated in 100 p1 of EX-CELL. Bioassays were performed as described below.
Prior to adding ligands, 5 pl of 21 nM melatonin in EX-CELL (1 nM final concentration) was added to each well to induce pigment aggregation. The cells were incubated in the dark in the presence of melatonin for 2 h then drugs were added to the wells in 5-p1 aliquots at 22 times their final concentration under a red light in the dark. Drug addition was performed in this manner in order to avoid pigment dispersion due to photostimulation. All drug solutions were prepared in EX-CELL and contained 1 nM melatonin. When preparing antagonists, the solutions also contained bombesin at its ECW value (0.2 nM). Peptide solutions were prepared on the day of assay from anhydrous peptide stocks or from frozen concentrated stock solutions in water that were stored at -20 "C for up to 1 month. Concentrated peptide solutions in water were aliquoted and used only once. When preparing antagonist stock solutions, the peptide content was assumed to be 70%. Melatonin was prepared from a 10 mM stock solution in ethanol stored at -20 "C. TPA was prepared as a concentrated stock solution in ethanol and diluted accordingly with EX-CELL. The final concentration of ethanol used in the assays was always less than 1 mM. Concentrations of ethanol less than or equal to 10 mM do not initiate pigment dispersion (19). Concentrated peptides were occasionally prepared in dimethyl sulfoxide but care was taken to ensure that the final concentration of dimethyl sulfoxide was less than or equal to 1.4 mM (0.01%, v/v) for the assays. Concentrations of dimethyl sulfoxide above 1.4 mM initiate pigment dispersion in a dose-dependent manner (data not shown).
Phototransmission was measured at 620 nm using a 340 ATTC microtiter plate reader (SLT Lab Instruments) or at 690 nm using a GCSS reader (SLT Lab Instruments). Transmission readings were taken 2 h after adding 1 nM melatonin (Ti). Drugs were immediately added and additional readings were made at various time points as specified in the text (T,). Measurements were made using the agglutination mode of the SOFT 2000 program (SLT Lab Instruments) (7) or the General Cell Screening System Version 1.0d6 or 1.0d9 (SLT Lab Instruments). When using the General Cell Screening System Version 1.0d6 (SLT Lab Instruments) the agglutination mode acquired 40 separate transmission measurements for each well of which the first and last five were discarded. The remaining 30 transmission readings were averaged and taken as an individual value. Data was directly transferred from General Cell Screening System to Microsoft Excel software for reduction.
Data was curve fitted with the equation for pK, determinations (see below) described by the nonlinear regression program Enzfitter (Biosoft), minimum plateau value of y, and ymaX = maximum plateau value of y. The nonweighted analyses were performed using the software program KaleidaGraph (Synergy Software). The error listed for each ECSO and ICs, value was determined by dividing by 2 the difference between the minimum and maximum anti-log,, (ECm or 1'260) value calculated from the standard error for each.
Cyclic A M P Quuntitation-Cyclic AMP studies were performed essentially as described by Daniolos et al. (9). Cells were electroporated with the appropriate vector (either pcDNAI/NeoBR or pON260) and seeded in 35-mm tissue culture dishes (Falcon) to an approximate density of 170,000 cells/dish. The cells were allowed to attach to the dish for at least 1.5 h before the medium was removed by aspiration and replaced with 2 ml of conditioned growth medium. After incubation at 27 "C for 48 h, the medium was removed by aspiration and the cells washed with 70% PBS (2 ml). The cells were then incubated overnight in EX-CELL (1.5 ml) and assayed the next day. A working concentration of 1 nM melatonin (15 pl of 100 nM melatonin in 70% PBS) was added to each dish to induce pigment aggregation. After incubating the cells in the dark for 30 min, bombesin or a-MSH was added at various concentrations and incubated for an additional 30 min. Drugs were prepared in 70% PBS at 100-fold the working concentration. The medium was then removed and cAMP extracted by adding 5 % (v/v) trichloroacetic acid (0.9 ml) followed by incubation at 4 "C for 30 min. The trichloroacetic acid was transferred to a polypropylene tube (15 ml, Falcon) and the cells washed with an additional 0.9 ml of 5% (v/v) trichloroacetic acid. The extracts were combined and extracted with water-saturated ether (3 X 10 ml). The samples were frozen at -20 "C and lyophilized to dryness. The pellets were redissolved in 1 ml of sodium acetate buffer (pH 6.2, Rianen Assay System, Du Pont) and the procedure for nonacylated cAMP samples was followed (Rainen Assay System, Du Pont). Radiolabeled iodine measurements were performed with a y-radiation counter (Beckman). Protein concentrations were determined relative to bovine serum albumin (fraction V; Sigma) using the Bio-Rad Protein Assay (Bio-Rad) following resuspension of the trichloroacetic acidextracted cells in 0.5 ml of 0.1 N NaOH/35-mm dish.
PHIPIP Labeling and Stimulation of Melanophore Cells-For experiments involving stimulation of [3H]PIP radiolabeled melanophores, cells were transfected with pJG3.6 or pJG3.6BR via electroporation as described earlier. Cells from three to four individual electroporations were pooled and equally proportioned into the same number of 24-well plates as there were electroporations (1.5 ml/well).
Approximately 1 X lo6 cells were plated per well. Three hours later, 1.2 ml of the medium was carefully removed by pipet and replaced with conditioned growth medium (2.0 ml). Two days later, the medium was removed and the cells were rinsed with EX-CELL (2.0 ml). 500 pl of the same medium containing my0-[2-~H]inositol (100 pCi/ml) was then added to each well. After 36 h of radiolabeling, the medium was removed, the cells were rinsed with fresh serum-free medium lacking my0-[2-~H]inositol(l ml) and replaced with the same medium (500 pl) containing 1 nM melatonin. The cells were incubated for 30 min in the dark. The medium was then removed and fresh medium (500 p1) containing melatonin (1 nM) with or without bornbesin (100 nM) was added, followed by incubation for 30 s in the dark. The reactions were quenched as described by Menniti et al. (20) and Sulpice et al. (21). The medium was quickly removed and an ice-cold solution (200 pl) of 6% perchloric acid (w/v) containing 500 pg/ml phytic acid was added to extract the inositol-containing metabolites.
Each plate was incubated at 4 "C for 20 min after which the acidic solution was transferred to a microcentrifuge tube and neutralized by adding a water-saturated KHC03 solution (45 pl). Once the evolution of carbon dioxide had dissipated, the solution was mixed thoroughly with a vortex (pH approximately 7.5 after neutralization) and centrifuged for 1 min in a microcentrifuge at maximum speed (15,000 X g). The aqueous layer was transferred to a new microcentrifuge tube, briefly centrifuged at 15,000 X g, and injected onto the HPLC.
Separation and Identification of Inositol Phosphates by HPLC-Resolution of the inositol phosphates was performed by anion-exchange chromatography using a Pharmacia Smart System. The procedures were essentially those described by Balla et al. In the text (see Fig. 2 and Table I), the identifiedpeaks are listed as di-or tri-inositol phosphates even though the peak listed as IP, coeluted with [3H]1,4-IP2 and in both controls the peak labeled as IP3 coeluted with [3H]1,4,5-IP3. The lack of more detailed study precluded absolute identification of the peaks because of the potential for coelution of other untested isomers.

TPA Induces Dose-dependent Pigment Dispersion within
Melanophores from Xenopus laeuis-As a first step to determine whether outward melanosome movement within melanophores might be induced independently of a rise in cAMP concentration, TPA, a potent activator of protein kinase C, was applied to the cells. To monitor any potential pigment movement, cells were seeded into a 96-well microplate at a concentration of approximately 15,000 cells/well and grown for 3 days. Melatonin was then added to the wells such that the pigment cells were exposed to a 1 nM concentration of the indole for 2 h. After melatonin treatment, during which the cells aggregated their pigment, TPA was added such that cells were exposed to concentrations ranging from 0.1 to 1000 nM.
A total of 11 concentrations of TPA were applied to quadruplicate wells. Immediately following the addition of TPA, and at 17 subsequent time points over the course of 2 h, the ability of the wells to allow transmission of light at 620 nm was determined with a microplate reader. Fig. 1A demonstrates the results of plotting the acquired data according to the formula 1-( T//Ti) versus time, where Ti is the initial measured phototransmission immediately before adding TPA and T, is that at selected subsequent time points (7). Pigment dispersion within melanophores and concomitant darkening within wells results in a decrease in Tf compared with Ti and is displayed as an upward displacement along the y axis. Beginning at 10 nM, TPA induces pigment dispersion which remains constant over the several hour duration of the experiment. Except for 1 PM TPA, the greatest concentration of the phorbol ester to which the cells were exposed, the higher the concentration of drug used to treat the cells, the greater their overall pigment dispersion. While 1 PM TPA causes a more rapid initial rate of cell darkening than any of the lower concentrations of phorhol est,er tested, the curve plateaus at a lower level than that engendered by 100 nM. At the present time, the reason for this reproducible effect is not known although it is possible t,hat 1 PM T P A is somewhat toxic to t h e cells.
To directly demonstrate the visual equivalent of the data obtained using the microtiter plate reader, the 96-well plate used in the experiment was fixed with 70% ethanol 2 h after treatment with TPA and Fig. 1R displays a photograph of four rows of the plate. From left to right, the wells in each row were exposed to increasing concentrations of T P A , while all wells in any given column were treated identically. Taken together, the four TPA-treated rows provide an "optical" doseresponse for the ahility of T P A t,o induce pigment dispersion within melanophores at 2 h. The 2-h data points displayed in Fig. 1A represent t.his information as interpreted by the microplate reader just prior to cell fixation.
The dose-response curve relating melanosome dispersion in a populat,ion of melanophores and the log concentration of TPA to which they were exposed can he fit to a simple hyperholic function. .'' The cells were extrarted to recover the inositol phosph;ttrs a n d the. extracts analyzed hy HI'1,C. Each fraction di.;playrrl in the thr chromatograms represent the mean n t t h c s c . r )~~n~s / n~i n / l r : t c . t i~~n ( I ! triplicate runs.

Comparison of the effects of bombesin treatment on inositol phosphate production in melanophores either transiently expressing the SBR or electroporated with the control plasmid pJG3.6
Cells electroporated with pJG3.6BR or pJG3.6 were plated into 24well plates, and after 1 day of growth the melanophores were labeled for 36 h with my~-[Z-~H]inositol in EX-CELL. Three days after electroporation, the media was removed, the cells washed with EX-CELL and aggregated with 1 nM melatonin for 30 min. After exposure for 30 s with 1 nM melatonin with or without 100 nM bombesin, the inositol phosphates were extracted and submitted to HPLC for separation as described under "Experimental Procedures." Only IP, and IP3 levels were measured. The presented results are mean values of triplicate experiments k S.E. Recombinant Melanophores Expressing SBRs Disperse Their Pigment in Response to Bombesin and Related Agonist Peptides in a Dose-dependent Manner- Fig. 3A demonstrates the results of applying different concentrations of bombesin and three related SBR peptide agonists, GRP18-27, litorin, and neuromedin B, to SBR-expressing melanophores. For all of the agonists, pigment dispersion reached a maximum after approximately 30 min and remained constant for the duration of the experiment. As with TPA before, the dose-response relationships between concentrations of all four peptides and pigment dispersion in SBR expressing melanophores can be described by a simple hyperbolic function. All of the peptides follow established rank order potencies for their ability to stimulate the SBR (23). For comparison, the figure also provides the results of applying the same peptides to wild type cells (Fig. 3B), and reveals no signals above background. The data shown in Fig. 3A was used to generate EC50 values (Table  11).
Results in Fig. 4 reveal that [Le~'~,\I113-14]Bn is a partial agonist with respect to pigment dispersion in melanophores transiently expressing the SBR but does not induce pigment dispersion in wild type melanophores. With an EC5,, of 13 nM (Table 11) methyl ester (25), to inhibit bombesin-induced pigment dispersion. Neither peptide induced pigment dispersion when applied to wild type cells (Fig. 5B). The experiments were performed by initially incubating the melanophores in 1 nM melatonin for 2 h to preset their melanosomes in a pigment-aggregated state. Next, the cells were simultaneously treated with 0.2 nM bombesin, essentially the EC50 value for bombesin as determined above, plus different concentrations of the antagonist peptides for 30 min. As with the application of agonists to cause pigment dispersion, the dose-response relationships for the two antagonists can be described by the same hyperbolic function  (Table 11), both peptides are potent antagonists for the SBR when it is expressed in melanophores.

DICUSSION
Initially, two questions arose, given the result that the protein kinase C activator, TPA, induced pigment dispersion.
Could pigment dispersion be induced in the absence of a rise  The displayed data was generated from recombinant cells derived from one set of electroporations to ensure that the observed differences in maximal response was not due to differential transfection efficiency. The day before the assay, the cells were not rinsed with EX-CELL before incubation in serum-free medium overnight. Data was derived by determining the agonist-induced change in phototransmission through cells. Each point in the graph is the mean from quadruplicate samples with error bars representing the corresponding standard errors of the mean.
in intracellular CAMP? And if so, because TPA is not a natural way of stimulating cells, could a more normal means, such as activation of a cell surface receptor that did not induce adenylate cyclase, nonetheless produce cell darkening? Because no endogenous receptors in frog melanophores were known which function via protein kinase C, cells were transfected with a plasmid containing cDNA coding for the Swiss 3T3 murine bombesin/gastrin-releasing peptide receptor (SBR). In its native environment, the bombesin receptor (BR) couples via a guanine nucleotide-binding protein (26-31) to phospholipase C. Bombesin stimulation triggers enhanced phosphoinositide metabolism, including the rapid formation of 1,4,5-IP3 (20, 32-34), mobilization of internal Ca2+ stores (32, 35), and activation of protein kinase C (36-39). When the receptor is expressed in novel environments such as frog oocytes, it retains its specificity for this pathway (14,40, 41).
Results from this study demonstrate that the murine SBR can be expressed in frog melanophores and that its stimula- tion with bombesin leads to the rapid formation of intracellular inositol phosphates, presumably catalyzed by phospholipase C. By definition, there must be a concomitant production of DAG. However, bombesin stimulation does not induce a rise in intracellular CAMP. A similar observation was reported for a clonal rat pituitary tumor cell line (GH4Cl) containing an endogenous BR. In this instance as well, GH4Cl cells stimulated with bombesin did not increase cellular cAMP levels (42,43).
The SBR displays appropriate pharmacological behavior when it is expressed in melanophores based on the characteristics for the two distinct BR subtypes. Two general classes of BR have been identified (44) and they can be differentiated on the basis of ligand binding and biological potencies (23, 40,[45][46][47]. One class of receptors, the NMB-R, has been found in rat esophageal muscularis mucosa, gastric smooth muscle cells, and the central nervous system. These receptors have a 10-20-fold greater binding affinity for neuromedin B than for GRP18-2, and they show little affinity for the BR antagonist [~-Phe']bombesin~~3 ethyl ester. The physiological potencies of the agonists mirror their binding affinities towards the NMB-R. The second class of bombesin receptor, the GRP-R, are found in Swiss 3T3 cells, pancreatic acini, rat gastric muscle strips, and guinea pig gastric smooth muscle cells. Compared to NMB-R, these receptors display greater binding affinities for GRP18-27 and poorer abilities to bind neuromedin B. When GRP-R-stimulated amylase release is measured, bombesin, litorin, and GRP18-27 are found to be approximately 10-20 times as potent as neuromedin B. Also, the antagonist [~-P h e ' ] b o m b e s i n~~~ ethyl ester is a potent inhibitor of the GRP-R. As measured by agonist-induced pigment dispersion in melanophores transiently expressing the SBR, bombesin, litorin, and GRPls-27 are approximately equipotent agonists, whereas neuromedin B is about 8-fold less effective (Table 11) The finding that [Le~'~-\k(CH~NH)-Leu'~]bombesin ([Le~'~,Bn) is a partial agonist for the SBR when it is expressed in frog melanophores was unexpected based on previous literature (Fig. 4). [Leu14,Bn is the first reported BR antagonist with a binding affinity of less than 100 nM (48). It has a K, of 65 nM for the SBR in Swiss 3T3 murine embryonal fibroblasts and has been shown to inhibit the growth of these cells as measured by its ability to prevent bombesin-stimulated incorporation of [3H]thymidine (IC50 = 18 nM) (49). Based on these findings, [Le~'~,\k13-14]Bn was expected to antagonize bombesin-induced pigment dispersion. However, Spindel etal. (41) reports that although [Leul4,\k13-14lBn shows substantial antagonist activity in oocytes expressing the SBR, it also exhibited partial agonist activity. In this particular study, bombesin stimulation was assayed by measuring light emission in oocytes that had been coinjected with RNA coding for the SBR and the calcium photoprotein aequorin. Although the possibility exists that [Leu1*,V13-14]-Bn or the receptor behaves somewhat differently in Swiss 3T3 cells compared with frog melanophores, the discrepancy may reflect the distinct assay methods and not the different cellular environments.
Receptor number and assay method probably influence measurements of EC50 and IC50. For example, the melanophore pigment dispersion system provides an EC5o value for bombesin of 0.2 nM and a minimum dose of 10 nM is required to observe a maximal biological response. Meanwhile, measurements of bombesin-induced amylase release from rat and guinea pig pancreatic acini or [3H]thymidine uptake into Swiss 3T3 cells show values of about 0.2 and 1 nM, respectively (25,44,48,50). Furthermore, in the latter assay, 3 nM bombesin provides a maximum biological response. Also, the melanophore assay reveals t h a t [~-P h e ' ] B n~~3 methyl ester has an IC50 of 17 nM while both classic assays show ICW values near 1 nM (23-25). The results of the different assays can be affected by factors such as the numbers of BRs expressed per cell and the type of cellular response being measured following the addition of chemicals. Regardless of the differences in the absolute EC50 and IC50 values seen with the three assays, the rank order potencies of the various agonists and antagonists are comparable.
It has been demonstrated that pigment dispersion in frog melanophores can be mediated by a CAMP-independent pathway. This alternative mechanism appears to utilize DAG as the second messenger for three reasons. First, TPA, a potent activator of protein-kinase C causes dose-dependent pigment dispersion. Second, when bombesin was added to recombinant cells expressing SBR, IP3 was produced while intracellular cAMP levels remained constant and pigment dispersion occurred. In addition, the substance P and thyrotropin-releasing hormone (TRH) receptors, which, like the SBR, activate phospholipase C following exposure to agonists (51,52), were expressed in melanophores. Substance P and TRH induced pigment dispersion with EC5o values of 3 f 1 and 0.09 f 0.03 nM, respectively, when applied to recombinant cells expressing the appropriate receptors (data not shown). Since DAG is a mandatory coproduct of 1,4,5-IP3 resulting from the cleavage of PIP2, it appears that DAG is the second messenger responsible for the induction of pigment movement following SBR stimulation. Third, 1,4,5-IP3, which typically functions to elevate intracellular calcium, does not appear to play a significant role in inducing centrifugal pigment movement. If it were a contributing factor, then raising intracellular calcium concentrations by treating the melanophores with the ionophore A23187 might be expected to induce pigment dispersion. When the cells were exposed to up to 10 p~ of the Caz+ ionophore, there was no observable dispersion of melanosomes (data not shown). Support for this conclusion comes from a recent publication in which it has been demonstrated that Ca2+ is not involved in pigment vesicle movement in fish melanophores (53). In summary, receptor-mediated DAG generation leads to pigment dispersion in a manner that appears to be as closely controlled as that accomplished via receptormediated cAMP production. This suggests that frog melanophores may have endogenous receptors that function via DAG to control the state of pigment disposition, and experiments to address this issue are in progress.
Finally, the ability of melanophores to disperse their pigment in response to the accumulation of DAG should provide a powerful tool for investigations into ligand-receptor interactions. Beside its utility for basic research, the assay described here may be useful for the rapid screening of drugs for their abilities to functionally interact with specific Gprotein-coupled receptors that activate phospholipase C.