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Stimulation of Intracellular Free Calcium in GH3 Cells by 3-Melanocyte-Stimulating Hormone. Involvement of a Novel Melanocortin Receptor?

Laboratory of Cell Pharmacology, University of Leuven, Medical School, Campus Gasthuisberg, B-3000 Leuven, Belgium

Address all correspondence and requests for reprints to: Prof. Carl Denef, Laboratory of Cell Pharmacology, University of Leuven, Medical School, Campus Gasthuisberg (O&N), B-3000 Leuven, Belgium. E-mail: carl.denef{at}med.kuleuven.ac.be

	Abstract

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The melanocortin (MC) 3MSH is a peptide that can be generated from the N-terminal domain of POMC and is believed to signal through the MC3 receptor. We recently showed that it induces a sustained rise in intracellular free calcium levels ([Ca²⁺]_i) in a subpopulation of pituitary cells, particularly in the lactosomatotroph lineage. In the present study we report that 3MSH and some analogs increase [Ca²⁺]_i in the GH- and PRL-secreting GH3 cell line and evaluate on the basis of pharmacological experiments and gene expression studies which MC receptor may be involved.

A dose as low as 1 pM 3MSH induced an oscillating [Ca²⁺]_i increase in a significant percentage of GH3 cells. Increasing the dose recruited an increasing number of responding cells; a maximum was reached at 0.1 nM. 2MSH, MSH, and NDP-MSH displayed a similar effect. SHU9119, an MC3 and MC4 receptor antagonist, and an MC5 receptor agonist, did not affect the number of cells showing a [Ca²⁺]_i rise in response to 3MSH. SHU9119 had also no effect when added alone. MTII, a potent synthetic agonist of the MC3, MC4, and MC5 receptor as well as an N-terminally extended recombinant analog of 3MSH showed low potency in increasing [Ca²⁺]_i in GH3 cells, but high potency in stimulating cAMP accumulation in HEK 293 cells stably transfected with the MC3 receptor. In contrast, a peptide corresponding to the 2MSH sequence of POMC-A of Acipenser transmontanus increased [Ca²⁺]_i in GH3 cells, but was about 50 times less potent than 2- or 3MSH in stimulating cAMP accumulation in the MC3 receptor expressing HEK 293 cells. By means of RT-PCR performed on a RNA extract from GH3 cells, the messenger RNA of the MC2, MC3, and MC4 receptor was undetectable, but messenger RNA of the MC5 receptor was clearly present.

These data suggest that the GH3 cell line does not mediate the effect of 3MSH through the MC3 receptor. The involvement of the MC5 receptor is unlikely, but cannot definitely be excluded. The findings animate the hypothesis that there exists a second, hitherto unidentified, MC receptor that displays high affinity for 3MSH.

	Introduction

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3 MSH IS A peptide that can be generated from the N-terminal domain of POMC and further processed to 2- and 1MSH (1). In contrast to MSH, the biological role of MSH peptides is not clearly established, although several biological actions have been reported in the brain, adrenal gland, kidney, and the cardiovascular system (for review see Refs. 2 and 3). We recently showed that 3MSH is biologically active in the anterior pituitary of immature rats, exerting a mitogenic effect on lactotrophs, somatotrophs, and thyrotrophs (4). 3MSH also induces a sustained rise in the intracellular free calcium concentration ([Ca²⁺]_i) in 15% of dispersed pituitary cells from immature rats (5). Within these responsive cells, 53% showed GH immunoreactivity (ir), 12% showed PRL ir, 2% showed TSH-ß ir, 5% showed LH-ß ir, and 10% showed ACTH ir, whereas 18% did not express any hormone to a detectable level (6).

MSH peptides act through specific membrane receptors known as melanocortin (MC) receptors, which are members of the family of G protein-coupled receptors, using elevation of cAMP as a signal transduction system (7, 8). Five MC receptor subtypes have been cloned. The MC1 receptor located in melanocytes and leukocytes has a role in pigmentation and inflammation (9). The MC2 receptor is the classical ACTH receptor of the adrenal gland (10). The MC3 and MC4 receptors are mainly expressed in the central nervous system (11). No clear-cut function of the MC3 receptor, which is also expressed in gut, heart, and placenta, has been defined yet, whereas the MC4 receptor is related to control of weight homeostasis (12, 13, 14). The MC5 receptor is expressed in various peripheral tissues (8, 15) and is believed to participate in exocrine gland regulation (16).

The only known receptor with high affinity for the MSH peptides is the MC3 receptor (2, 7, 17). However, several observations on the action of MSH peptides in the cardiovascular system suggest the existence of other MSH receptors (18, 19). Recently, we showed the expression of MC3 receptor messenger RNA (mRNA) in the anterior pituitary of immature rats (5), but SHU9119, an MC3 receptor antagonist (13, 20), blocked the effect of 3MSH on [Ca²⁺]_i in less than 50% of the responsive cells (5). The cell types in which SHU9119 was ineffective were PRL-, GH-, and TSH-immunoreactive cells (6), suggesting that the [Ca²⁺]_i responses induced by 3MSH in the latter pituitary cell types may not be mediated by the MC3 receptor.

In the present study we explored whether GH3 cells, a pituitary cell line representative for lactotrophs and somatotrophs, would display [Ca²⁺]_i responses to 3MSH, and if so, whether these [Ca²⁺]_i responses would also be mediated by a receptor pharmacologically distinct from the MC3 receptor. Finally, it was tested by RT-PCR which MC receptors are expressed, at least at the mRNA level, in the GH3 cell line.

	Materials and Methods

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Peptides and pharmacological reagents
3MSH, 2MSH, MSH (acetylated), and [Nle⁴,D-Phe⁷]MSH were purchased from Peninsula Laboratories, Inc., Europe (Merseyside, UK). Ac-Nle⁴-cyclo-[Asp⁵,(D-Nal²)⁷,Lys¹⁰]MSH-(4–10)NH₂ (SHU9119) was obtained from Neosystems Laboratoire (Strasbourg, France), and Ac-Nle⁴-cyclo-[Asp⁵,D-Phe(pF)⁷,Lys¹⁰]MSH-(4–10)-NH₂ (MTII) (21) was obtained from Bachem AG (Bubendorf, Switzerland). An N-terminal-extended form of 3MSH, designated Ala^2,8,20,24-POMC-(1–74), in which the cysteines at positions 2, 8, 20, and 24 were mutated to Ala, thus eliminating the two naturally occurring cysteine bridges, was produced in our laboratory by means of the pGEX-4T-1 expression vector (Amersham Pharmacia Biotech, Uppsala, Sweden) in BL21 protease-deficient Escherichia coli cells, purified, and characterized as described previously (22). Mass spectrometry and N-terminal amino acid sequencing showed the authenticity of the product. A 2MSH analog based on the POMC-A sequence of the white sturgeon Acipenser transmontanus (Tyr-Val-Met-Ser-His-Phe-His-Trp-Asn-Thr-Phe-Gly) was synthesized by Sigma-Genosys (Cambridge, UK). Peptides were prepared as stock solutions at a concentration of 0.1 mM in 0.1% crystalline BSA (Serva, Heidelberg, Germany). HBSS was purchased from Life Technologies, Inc., Europe (Paisley, UK), and pluronic F127 and fluo-3 acetoxymethyl ester (fluo-3/AM) were obtained from Molecular Probes, Inc. (Eugene, OR). All of these reagents were prepared as stock solutions and maintained at -25 C. All reagents were analytical grade.

Cell culture
The GH₃ cell line was obtained from American Type Culture Collection (CCL 82.1; Manassas, VA). At the start of this study cells were cultured in DMEM (Life Technologies, Inc.) supplemented with 10% FCS (Life Technologies, Inc.). In later experiments it was found that certain serum batches attenuated the effect of 3MSH, which was avoided when cells were cultured in defined culture medium (mixture of DMEM and F12 medium; Life Technologies, Inc.) as described previously (23) supplemented with 1% FCS.

Cultures were maintained in a water-saturated incubator (1.5% CO₂-98.5% air) at 37 C. Subcultures were made each week, and medium was renewed every 3 days. Twenty-four hours before an experiment, cells were trypsinized and seeded on a Lab-Tek chambered slide (1.8 cm²/chamber; 250,000 cells/chamber; Nunc, Roskilde, Denmark), coated with polyornythine (0.004%; Sigma-Genosys) and maintained in defined culture medium without serum supplement.

Intracellular free calcium measurements
[Ca²⁺]_i changes in response to the different peptides were tested 24 h after trypsinization. [Ca²⁺]_i was recorded by a video imaging system allowing the simultaneous study of 30–40 cells/field. Imaging and measurement of [Ca²⁺]_i were performed by means of the fluorescent dye fluo-3 (24) as previously described (25). Briefly, cells were incubated for 15 min at 37 C with 10 µM fluo-3/AM and 0.02% pluronic F127 in HBSS. After loading, cells were rinsed once with and kept in HBSS (bath medium). Peptides were directly diluted in bath medium at the concentration indicated in the experiments. Recording of the fluorescence was performed at room temperature using a Meridian Insight confocal microscope (Okemos, MI) based on an Olympus Corp. IMT2 inverted microscope (Melville, NY) with a D-plan APOx100 (NA 1.25) oil immersion objective equipped for microspectrofluorometry. A light optical control image was obtained with a charge-coupled device camera fixed on the side of the microscope. The dye was excited at 488 nm using an argon ion laser (532 argon ion laser, Coherent, Palo Alto, CA), and the emitted light was recorded at 530 nm. The fluorescence was amplified using an image intensifier (Dage MTI, Michigan City, IN) and collected by a cooled charge-coupled device camera (Meridian Instruments, Lansing, MI). The video image obtained was recorded on high quality S-VHS videotape, equipped with a digital noise reduction board and a RS232 computer interface. From the VCR, the recorded images can be analyzed up to the video rate (25 images/sec). The video images were digitized (at 8-bit accuracy to yield 256 gray levels, with 768 x 512 pixels maximal spatial resolution) with a frame grabber board (Vision-EZ, Data Translation, Marlboro, MA) and transferred to the computer hard disk. A maximum of 200 images of up to 768 x 512 pixels were used per analysis. For a total recording time of 4 min, we collected one image every 1.18 sec. Subsequent image analysis was performed using in-house software. To obtain a continuous trace of mean fluorescence as a function of time, polygons were drawn over each recorded cell on the light-optical control picture. The average fluorescence value in each polygon was then calculated by adding all pixel fluorescence values (8 bits/pixel) and subsequently dividing this total amount by the number of pixels in the polygon, yielding a value between 0 and 255.

Fluo-3 was chosen as Ca²⁺ indicator because of its large optical signal, which allowed a very good signal to noise ratio in a single frame. The lack of emission or excitation spectral shift of fluo-3 on Ca²⁺ binding makes it difficult to calibrate fluorescence signals in terms of precise and absolute values of free [Ca²⁺]_i. Therefore, fluorescence signals are expressed as (F - F₀)/F₀ ratios, with F being the fluorescence during response to a peptide, and F₀ the minimal resting fluorescence before stimulation. Changes in normalized fluorescence are therefore indicative of changes in [Ca²⁺]_i values.

Part of the GH₃ cells displayed spontaneous [Ca²⁺]_i oscillations with variable frequency and amplitude. The mean amplitude of nonoscillating cells was 0.11. Spontaneous oscillations were defined as oscillations with amplitude greater then 0.25. In nonoscillating cells, a positive Ca²⁺ response was defined by the appearance of fluo-3 fluorescence oscillations with amplitude exceeding 0.25. In spontaneously oscillating cells, a positive Ca²⁺ response was defined by the appearance of fluorescence oscillations with an integrated fluorescence (expressed per min) exceeding the mean basal integrated fluorescence (expressed per min) and/or by the appearance of oscillations displaying an augmentation in frequency (Fig. 1). Integrated fluorescence, delay in the onset of response, and oscillation frequency of [Ca²⁺]_i were determined for each responsive cell.

View larger version (28K): [in this window] [in a new window]   Figure 1. Effect of 1 nM 3MSH on [Ca2+]i in GH3 cells. [Ca2+]i changes were studied by fluorescence microscopy using video imaging in fluo-3-loaded cells. 3MSH was applied by direct dilution in the bath medium (HBSS). A, Example of a responsive nonoscillating cell. B, Example of a responsive spontaneously oscillating cell.

RT-PCR
Total RNA from the GH₃ cell line, pituitary cells and brain of 14-day-old rats, adrenal gland from adult rats, and HEK293 cells stably transfected with the MC3 receptor were isolated by the guanidium thiocyanate-phenol-chloroform extraction procedure (26). After a deoxyribonuclease I (1 U/10 µl, 1 h at 25 C; Life Technologies, Inc.) treatment, to eliminate genomic DNA, 2.5 µg total RNA were reverse transcribed into cDNA at 42 C using random hexamer primers (Perkin-Elmer Corp., Branchburg, NJ) and Moloney leukemia virus reverse transcriptase (Perkin-Elmer Corp.) in a 20-µl final volume, followed by PCR, both as described below. To check for possible artifacts generated by amplification of remnants of genomic DNA, the RT-PCRs performed for detecting mRNA of MC receptors (MC receptor genes lack introns) (27, 28) were also performed on a nonreverse transcribed RNA sample (RT omitted).

PCR amplification
The PCR primers used to amplify the RT-generated hormone (GH and PRL) and MC receptor cDNAs were designed on the basis of established GenBank sequences with commercially available software (Genejockey II, Biosoft, Cambridge, UK). Primers were synthesized by Pharmacia Biotech. The forward and backward primers for GH cDNA (GenBank accession no. J00739 and V01239) were 5'-GCTGCAGACTCTCAGACTCCCTGG-3' (nucleotides 464–470 and 655–672) and 5'-GTCTCTGAGAAGCAGAACGCA-3' (nucleotides 1608–1587). This set yielded a product of 242 bp. The primers for PRL cDNA (GenBank accession no. J00760) were 5'-ACCATGAACAGCCAGGTGTCAG-3' (nucleotides 474–494) and 5'-CTTGTCTTCAGGAGTAGCTAG-3' (nucleotides 453–434) and yielded a product of 291 bp. The forward and backward primers for MC3 receptor cDNA (GenBank accession no. X70667) were 5'-ACATGCTGGTGAGCCTGTCCAA-3' (nucleotides 549–570) and 5'-CTAACCGTCATGATGCTGTGGTAAC-3' (nucleotides 774–750). This set yielded a product of 226 bp. The forward and backward primers for MC4 receptor cDNA (GenBank accession no. U67863) were 5'-TTTCATCTGTAGTCTGGCT-3' (nucleotides 389–408) and 5'-GAACGCCCGATACTGTGCAAGCT-3' (nucleotides 694–672). This set yielded a product of 305 bp. The forward and backward primers for MC5 receptor (cDNA GenBank accession no. L27081) were 5'-TTCTTTGTGGGCAGCCTAG-3' (nucleotides 469–487) and 5'-CAGGGCGTAGAAGATGGTGATGTAC-3' (nucleotides 696–672). This set yielded a product of 228 bp. As the sequence of rat MC2 receptor has not yet been determined, we choose the primers for MC2 receptor DNA based on sequence homologies between MC2 receptor DNA from mouse and hamster. The forward and backward primers for MC2 receptor cDNA (mouse, GenBank accession no. D31952; hamster, GenBank accession no. U71279) were 5'-CAGCAGGAAAAAATGAAGCATAT-3' (mouse, nucleotides 137–159; hamster, nucleotides 46–67) and 5'-ATGTCATCTGCTGTGCTTTC-3' (mouse, nucleotides 462–443; hamster, nucleotides 370–351), yielding a product of 325 bp.

PCR of the RT-generated cDNA was performed using a GeneAmp PCR System 2400 thermal cycler (Perkin-Elmer Corp.). The PCR mixture contained 1 µl reverse transcriptase template added to a mixture of (final concentrations) 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 4.5 mM MgCl₂, 200 µM of each deoxy-NTP, sense and antisense primer (1 µM of each), and 1 U AmpliTaq Gold (Perkin-Elmer Corp.) in a final volume of 25 µl. Conditions of DNA amplification were the same for all pairs of primers and included an initial denaturation step of 10 min at 95 C (which also activates the Gold variant of the Taq polymerase) and 40 cycles of 20 sec at 95 C, 20 sec at 60 C, 30 sec at 72 C, and finally 7 min at 72 C. Half of the PCR samples were electrophoresed in a 2% agarose gel and stained with ethidium bromide (0.5 µg/ml). To confirm the identity of the amplified products, restriction analysis was carried out on each PCR product using specific restriction enzymes (Life Technologies, Inc.) as described previously (6): HaeIII (different restriction profiles for GH and PRL cDNA PCR products), AvaII (MC3 receptor cDNA specific), SsP1 (MC4 receptor cDNA specific), and MboI (MC5 receptor cDNA specific).

Data analysis
All experiments were repeated at least three times. Values were expressed as the mean ± SE of several independent experiments and compared using ANOVA (NCSS, Statistical Solutions Ltd., Cork, Ireland). Parameters of [Ca²⁺]_i oscillations induced by the peptides (integrated fluorescence, frequency of oscillations, and delay) were compared with those recorded in resting conditions. Statistical analysis was performed using one- or two-way ANOVA. The Tukey-Kramer test was used whenever the ANOVA test indicated a significant difference. Curve fitting and EC₅₀ value calculation, if applicable, were performed with the statistical software packets Curve Expert 1.3 or Prism (GraphPad Software, Inc., San Diego, CA). Differences were considered statistically significant at P < 0.05.

	Results

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Characterization of [Ca²⁺]_i in GH3 cells
To study the basal [Ca²⁺]_i activities in GH3 cells, more than 1000 cells were recorded in resting conditions. Sixty percent of the cells showed spontaneous basal [Ca²⁺]_i oscillations, whereas the remainder did not show any oscillation over a total recording time of 4 min. A small number of cells (10%) that were spontaneously inactive during the 1 min of recording of basal activity started to show spontaneous [Ca²⁺]_i oscillations at a particular time point during the next 3 min of recording. Therefore, in each experiment a control well with cells was recorded during these 3 min without adding the peptide under study. The data obtained were corrected for these false positive responders. There was no effect on [Ca²⁺]_i after application of the vehicle only (609 cells were tested in a number of randomly chosen experiments).

Effects of 3MSH on [Ca²⁺]_i in GH3 cells
Addition of 3MSH induced a sustained rise in [Ca²⁺]_i in spontaneously nonoscillating as well as in oscillating cells from picomolar doses. An example is shown in Fig. 1. At all doses used, about half of the responsive cells were nonoscillating. As shown in Table 1 there was a significant rise in the integrated fluorescence. In spontaneously oscillating cells the frequency of [Ca²⁺]_i oscillations also increased. There was no consistent relation observed between the 3MSH concentrations and the delay in the [Ca²⁺]_i responses, the integrated fluorescence values, and the frequency of the [Ca²⁺]_i oscillations, at least within the dose range used. In contrast, the dose of the peptide clearly influenced the number of cells that displayed increased [Ca²⁺]_i. As shown in Fig. 2 a concentration of 1 pM 3MSH induced a sustained increase in [Ca²⁺]_i in 14.2% of the analyzed cells, whereas 24.0% and 21.2% of the cells were responsive at 0.1 and 1 nM, respectively. At concentrations of 10 and 100 nM 3MSH, the number of responsive cells was reduced (12.9% and 9.8%, respectively) compared with that found with 1 nM. Curve-fitting showed a quadratic fit with r = 0.88, indicating a biphasic bell-shaped dose-response curve.

View this table: [in this window] [in a new window]   Table 1. Effects of different concentrations of 3MSH on the characteristics of the Ca2+ responses

View larger version (13K): [in this window] [in a new window]   Figure 2. Effects of different concentrations of 3MSH on the percentage of GH3 cells displaying [Ca2+]i responses to the peptide. In each experiment at least two concentrations were tested together and compared with vehicle. Values obtained with vehicle only (false positive responders) were subtracted from the values obtained after addition of peptides. Each point is expressed as the mean ± SEM of the total number of cells responding (spontaneously oscillating and nonoscillating cells combined). The number of independent experiments was 4, 3, 3, 7, 5, 13, and 5 for 0.1 pM, 1 pM, 10 pM, 100 pM, 1 nM, 10 nM, and 100 nM, respectively. By overall one-way ANOVA, P < 0.0001; multiple comparisons: #, different from the next value by P < 0.05 at least; *, different from the control value at P < 0.05; **, different from the control value at P < 0.01.

View larger version (16K): [in this window] [in a new window]   Figure 3. A, Effects of MSH, NDP-MSH, and Ala2,8,20,24-POMC-(1–74) on [Ca2+]i in GH3 cells, compared with that of 3MSH. B, Effect of MTII and Acipenser 2MSH on [Ca2+]i in GH3 cells compared with that of 2MSH (0.1 and 1 nM) and 3MSH (0.01 and 1 nM). In each experiment at least two concentrations were tested together and compared with vehicle. Values obtained with vehicle only (false positive responders) were subtracted from the values obtained after addition of peptides. Each point is expressed as the mean ± SEM of the total number of cells responding (spontaneously oscillating and nonoscillating cells combined). 3MSH was always tested together with the other analogs. Number of experiments: MSH, eight; NDP-MSH, four; MTII, seven; and Ala2,8,20,24-POMC-(1–74), seven. For Acipenser 2MSH, 0.1 and 1 nM were tested three times together with 2MSH; 0.01 and 100 nM were tested four times together with 3MSH. The total number of cells tested for each dose level ranged between 203 and 563. *, P < 0.05; **, P < 0.01 (vs. control). MSH (1 nM) is different from both 0.01 and 100 nM at P < 0.05. Acipenser 2MSH (0.01 nM) is different from both 1 and 100 nM at P < 0.05. 2MSH (0.1 nM) is different from Acipenser 2MSH (0.01, 1, and 100 nM). No statistically significant effects were found for MTII and Ala2,8,20,24-POMC-(1–74).

In a second experimental set-up, the effect of MTII (13, 20, 29), a potent agonist of the MC3 (EC₅₀, 0.19–0.27 nM) (13, 20) and MC4 receptor (EC₅₀, 0.019–0.057 nM) (13, 20) and a somewhat weaker agonist of the MC5 receptor (EC₅₀, 1.36 nM) (13), was studied. MTII had no significant effect on [Ca²⁺]_i at doses of 0.1, 1, 10, and 100 nM (Fig. 3B).

The Acipenser transmontanus 2MSH sequence Tyr-Val-Met-Ser-His-Phe-His-Trp-Asn-Thr-Phe-Gly was tested because chondrostean fish display a degenerated 3MSH sequence, including a mutation in the His-Phe-Arg-Trp core sequence (30), anticipating a low potency of the Acipenser peptide in MC3 receptor-mediated responses (31). Acipenser 2MSH was compared with mammalian 2MSH and 3MSH. Acipenser 2MSH was ineffective at 0.01 nM, but was significantly effective at 0.1 and 1 nM. At the latter doses it was only slightly less active (although not statistically significant) than the same doses of 2MSH tested in the same experiment (Fig. 3B). It is noteworthy that a maximal effect was reached at the same dose as that of 2MSH (or 3MSH compared with data in Fig. 2).

When cells were pretreated for 5 min with the MC3 receptor antagonist SHU9119 (0.1 and 1 µM) and then tested for [Ca²⁺]_i responses to 0.1 and 10 nM 3MSH, in the presence of the same amount of the antagonist, the percentage of cells showing [Ca²⁺]_i responses did not alter (Fig. 4). Treatment with 0.01, 1, or 10 nM SHU9119 (three independent experiments; data not shown) or with 1 µM SHU9119 alone (three independent experiments; Fig. 4) did not affect [Ca²⁺]_i in GH3 cells (tested immediately after application of the peptide).

View larger version (16K): [in this window] [in a new window]   Figure 4. Effect of the MC3 receptor antagonist SHU9119 on the [Ca2+]i responses induced by 3MSH. Values obtained with vehicle only (false positive responders) were subtracted from the values obtained after addition of peptides. Data are the mean ± SEM. A dose of 0.1 nM 3MSH with and without 100 nM SHU9119 was tested in three different experiments; a dose of 10 nM 3MSH with and without 1 µM SHU9119 was tested in four different experiments.The total number of cells tested for each dose level ranged between 214 and 421. No significant differences between [Ca2+]i responses in the presence and absence of SHU9119 were found.

Effect of 3MSH and analogs on cAMP levels in HEK 293 cells expressing the MC3 receptor and in CHO cells expressing the MC5 receptor
In the HEK 293 cells transfected with the MC3 receptor, 1 nM MTII caused a 10-fold increase in cAMP levels, as expected (Table 2). Ala^2,8,20,24-POMC-(1–74) was almost as potent as 3MSH in increasing cAMP accumulation; EC₅₀ values were 1.7 and 5.1 nM in two independent experiments (Table 2) vs. 0.07 and 1 nM for 3MSH (Table 3). In contrast Acipenser 2MSH was 17–94 times less potent than mammalian 3MSH (Table 3).

View this table: [in this window] [in a new window]   Table 2. Effects of 3MSH, 3MSH in the presence of a 100-fold excess of SHU9119, MTII, and Ala2,8,20,24-POMC-(1–74) on cAMP accumulation in HEK 293 cells stably transfected with the MC3 receptor

View this table: [in this window] [in a new window]   Table 3. Effect of Acipenser transmontanus 2MSH compared to that of mammalian 3MSH on cAMP accumulation in HEK 293 cells stably transfected with the MC3 receptor

As shown in Table 4, both 3MSH and Ala^2,8,20,24-POMC-(1–74) caused an increase in cAMP in CHO cells transfected with the MC5 receptor. 3MSH showed activity starting from a 3-nM dose, whereas Ala^2,8,20,24-POMC-(1–74) was more than 10 times less potent.

View this table: [in this window] [in a new window]   Table 4. Effect of Ala2,8,20,24-POMC-(1–74) compared to that of 3MSH on cAMP accumulation in CHO cells stably transfected with the MC5 receptor

View larger version (59K): [in this window] [in a new window]   Figure 5. MC receptor mRNA expression in the GH3 cell line as determined by RT-PCR. Total RNA of GH3 cells, normal pituitary cells, HEK 293 cells expressing MC3 receptor, rat brain, and adrenal were reverse transcribed, followed by PCR for the MC2 receptor (A; amplified fragment of 325 bp), the MC3 receptor (B; amplified fragments of 226 bp), MC4 receptor (C; 305 bp), and MC5 receptor (D; 228 bp), using specific primers for each receptor subtype as described in Materials and Methods. RNA from adrenal (Adr), normal rat pituitary cells (Pit), HEK293-MC3 receptor (HEK293), and rat brain (brain) and genomic DNA from GH3 cells (DNA) were used as positive controls, and H2O and samples without reverse transcriptase (-RT) were used as negative controls. Amplified fragments were resolved by 2% agarose gel electrophoresis and visualized by ethidium bromide staining. M, Molecular weight markers.

View larger version (85K): [in this window] [in a new window]   Figure 6. GH and PRL mRNA expression in the GH3 cells as determined by RT-PCR. Total RNA from GH3 cells was subjected to RT, followed by PCR for PRL and GH cDNA (amplified fragments of 291 and 242 bp, respectively) in the same reaction (duplex PCR), using rat-specific primers. Amplified fragments were resolved by agarose-gel electrophoresis and visualized by ethidium bromide staining (0.5 g/ml). RNA from normal pituitary cells (Pit) was used as a positive control; H2O and samples without reverse transcriptase (-RT) were used as negative controls. M, Molecular weight markers.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The data obtained from experiments with various agonists and antagonists of MC receptors strongly plead against mediation of the effect of 3MSH by the MC3 receptor. First, 3MSH induced an increase in [Ca²⁺]_i at doses several orders of magnitude beneath those reported in the classical cAMP stimulation test in cell lines stably transfected with the MC3 receptor (reviewed in Ref. 34 and also in the present study). The minimal dose effective on [Ca²⁺]_i in GH3 cells was as low as 1 pM, and the maximal effect was seen at 0.1 nM, whereas the EC₅₀ for stimulation of cAMP formation in the HEK 293 cells was 0.07–1 nM in our hands [reported to be 4 nM by others (11)]. Second, SHU9119, a competitive MC3 receptor antagonist (20) did not change the number of cells showing [Ca²⁺]_i responses to 3MSH at a dose sufficient to fully block the MC3 receptor [1000 times in excess of 3MSH (20)]. Third, MTII, a synthetic cyclic MSH analog (21) with potent agonist activity at the MC3 receptor transfected in heterologous cell lines [(EC₅₀ for cAMP accumulation, 0.19, 0.27, and 1.2 nM, depending on the report (13, 20, 35)], failed to induce an increase in [Ca²⁺]_i in GH3 cells at doses up to 100 nM, which is 3 orders of magnitude above the dose inducing a maximal effect of 3MSH in these cells. Fourth, Ala^2,8,20,24-POMC-(1–74), an analog of 3MSH N-terminally extended with more than 50 amino acid residues [i.e. POMC-(1–74), in which the cysteines at positions 2, 8, 20, and 24 are mutated to Ala] was highly effective in stimulating the MC3 receptor stably transfected in HEK 293 cells, but had no effect on [Ca²⁺]_i in GH3 cells, even at a dose 1000-fold higher than the maximally effective dose of 3MSH in the latter cells. In contrast, Acipenser 2MSH was a weak agonist in the cAMP assay, but was fairly effective on [Ca²⁺]_i in GH3 cells. Finally, by means of RT-PCR, no mRNA of the MC3 receptor was detectable in GH3 cells, although this mRNA was detected in brain and normal pituitary, as reported previously (5).

Does another known MC receptor mediate the [Ca²⁺]_i response to 3MSH? It is most unlikely that the MC4 receptor is involved. First, SHU9119, an even more potent antagonist of this receptor (20), was ineffective on 3MSH-induced [Ca²⁺]_i changes in GH3 cells, and MTII, also a potent agonist of the MC4 receptor [EC₅₀, 0.019, 0.057, or 0.17 nM, depending on the reported study (13, 20, 35)] was ineffective up to doses several orders of magnitude higher than the effective doses of 3MSH. Second, mRNA of the MC4 receptor could not be detected in GH3 cells. In contrast, MC5 receptor mRNA was found in GH3 cells (this study). The EC₅₀ for the effects of MSH peptides on cAMP levels through the MC5 receptor transfected in cell lines is in the middle nanomolar range (8, 28, 36, 37). In our hands, as tested for accumulation of cAMP in CHO cells stably transfected with the rat MC5 receptor, the EC₅₀ of 3MSH was 11.3 nM. Thus, it is reasonable to reflect on the hypothesis that the MC5 receptor may be involved in the 3MSH-induced [Ca²⁺]_i responses. However, several findings do not support this hypothesis. First, although the EC₅₀ value for the 3MSH effect in GH3 cells could not be exactly determined because of the bell-shaped dose-response curve, it appears to be about 3 orders of magnitude lower than that for 3MSH in the CHO cell line. Second, SHU9119 is a full agonist of the MC5 receptor (EC₅₀, 0.43–2.31 nM) (20, 38), but it did not cause [Ca²⁺]_i responses in GH3 cells at concentrations up to 1 µM. Third, MTII (21) is also a very potent agonist of the MC5 receptor (EC₅₀, 1.4–2.3 nM) (20, 35, 38), but it did not induce a significant increase in [Ca²⁺]_i at a dose up to 100 nM. Finally, Ala^2,8,20,24-POMC-(1–74) was 10 times less potent than 3MSH in the CHO MC5 receptor cell line, but was at least 1000 times less potent than 3MSH in GH3 cells (ineffective at 100 nM, whereas 3MSH was maximally effective at 0.1 nM). Because of its high potency on [Ca²⁺]_i, 3MSH is also unlikely to act through an MC1 or MC2 receptor (34). Furthermore, there was no evidence found for expression of the MC2 receptor in GH3 cells, and the inactivity of MTII in GH3 cells is not in accordance with the reported EC₅₀ value of MTII (in the subnanomolar range) at the MC1 receptor (20).

It should be realized, however, that the interaction of the MC5 receptor with agonists and antagonists has mainly been studied in cell lines overexpressing the receptor after transfection. One should bear in mind that the orders of potency of these agonists and antagonists at the MC5 receptor expressed in its natural microenvironment, such as in GH3 cells, may be different compared with those at the receptor transfected in heterologous cell lines. Therefore, we cannot exclude with certainty that 3MSH in GH3 cells acts on [Ca²⁺]_i through the MC5 receptor. However, if this were the case, the receptor would acquire a gain of function of some 3 orders of magnitude with respect to the natural agonist 3MSH and a loss of function of a similar magnitude with respect to several chemically distinct synthetic MC5 receptor agonists [MTII, SHU9119, and Ala^2,8,20,24-POMC-(1–74)]. These data are difficult to reconcile with the hypothesis that the [Ca²⁺]_i response to 3MSH in GH3 cells is mediated by the MC5 receptor. It is reasonable, therefore, to propose the hypothesis that there may exist a hitherto uncharacterized receptor with high affinity for MSH peptides.

It is interesting to note that the high pharmacological potency of 3MSH on this hypothetical novel receptor is not associated with selectivity, as MSH and NDP-MSH were also effective. The pharmacological characteristics of the 3MSH effect in GH3 cells are therefore different from those reported in the cardiovascular system, in which an increase in heart rate has been noticed with 2MSH, but not with 3MSH or MSH (39). In contrast, MTII and SHU9119, which both have a structurally constrained cyclic MSH core sequence (20, 21), act, respectively, as potent agonist and antagonist at the MC3 receptor in transfected HEK 293 cells, but not at the receptor mediating [Ca²⁺]_i changes in GH3 cells. Thus, constraining the MSH core sequence by cyclization does not affect interaction with the MC3 receptor, but seems to impair interaction with the hypothetical receptor for 3MSH in GH3 cells. A differential influence of structural restraint was also seen by N-terminal extension of 3MSH in Ala^2,8,20,24-POMC-(1–74). Such extension was deleterious for the action at the hypothetical novel 3MSH receptor, but had minimal influence on the action at the MC3 receptor.

Chondrostean fish, like Acipenser transmontanus (white sturgeon), have a duplicated POMC gene (POMC A and POMC B) (40). POMC B has a conserved MSH core sequence, but in POMC A the third residue of the MSH core sequence, His-Phe-Arg-Trp, is mutated to His. It has therefore been suggested that the MSH sequence is degenerated in chondrostean fish (30). It is well known that single amino acid substitutions in the core sequence of MSH peptides cause a dramatic decrease in affinity and activity of these peptides at the MC receptors (31, 41). Acipenser 2MSH was indeed about 50 times less potent at the MC3 receptor in HEK 293 cells compared with mammalian 2MSH. However, the finding of a fairly effective action of Acipenser 2MSH in GH3 cells (maximal effect reached at the same dose as that of 2MSH or 3MSH) suggests that the core sequence for the effect on [Ca²⁺]_i is different from that of MSH peptides at the known MC receptors. Moreover, these data suggest that the MSH sequence from the POMC A gene of Acipenser may not be corrupt for all its biological actions.

It is surprising that only about 20% of the GH3 cells responded to 3MSH. It is known that GH3 cells are functionally heterogeneous (42, 43). This functional heterogeneity could be translated in heterogeneity in receptor expression or action. Moreover, electrophysiological experiments have shown that only about 50% of cultured GH3 cells are excitable, and that during certain cell cycle phases cells are electrically inactive and retain secretion rates at their basal level after treatment with TRH (44). As during standard culture the GH3 cell population is a mixture of cells in different stages of the cell cycle, it can be expected that [Ca²⁺]_i changes in response to 3MSH may not occur in all cells. In fact, TRH induced a [Ca²⁺]_i response in only about 20% of the cells.

An intriguing finding was that the dose-response curve of 3MSH was biphasic. Increasing doses in the picomolar range recruited an increasing number of cells displaying a [Ca²⁺]_i rise, whereas above 1 nM the number of responding cells markedly declined. It is possible that 3MSH activates two signal transduction pathways with opposite effects, together creating a bell-shaped dose-response curve. Such a phenomenon was reported in Hepa cells transfected with the human MC3 receptor (45). Bell-shaped dose response relations are also characteristic of trophic effects of melanocortins on cultured neurons in vitro as well as on neurite outgrowth in vivo (46, 47, 48). Although the significance and mechanism of the bell-shaped dose response remain unclear, it can be speculated that the [Ca²⁺]_i response under physiological conditions occurs only within a certain window of 3MSH concentrations. Such a response characteristic is reminiscent of that of morphogen gradients that induce different cellular responses and cell fates at different concentrations (49). Thus, in a physiological environment 3MSH may act on [Ca²⁺]_i-dependent processes not in cells in the immediate vicinity of the site of peptide secretion, but on remote cells where the concentration has fallen to lower levels as a consequence of diffusion.

In conclusion, the present data strongly suggest that the action of MSH peptides on [Ca²⁺]_i in the GH3 lactosomatotrope cell line is not mediated by the MC3 receptor. Although the MC5 receptor cannot definitely be excluded as the responsible receptor, mediation of the [Ca²⁺]_i response by this receptor seems unlikely. The data encourage further testing of the hypothesis that a hitherto unidentified MC receptor, displaying high affinity for 3MSH, may exist. We previously reported that SHU9119 failed to block the [Ca²⁺]_i response to 3MSH in GH and PRL cells in normal rat pituitary (6). Possibly, normal lactotrophs and somatotrophs also express the hypothetical receptor presently found in GH3 cells. The use of the GH3 cell line will allow us to further identify the receptor involved in the actions of 3MSH in these cells and possibly in normal pituitary cells.

	Footnotes

 
¹ Supported by a fellowship from the Flemish Institute for the promotion of Scientific-Technological Research in Industry.

Received April 27, 2000.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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