Introduction

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The mammalian Bn-related peptides NMB and GRP mediate a diversity of biological responses, including thermoregulation, satiety, control of circadian rhythm, stimulation of pancreatic secretion and stimulation of GI hormone release (Tache et al., 1988). In addition, these peptides exhibit potent developmental effects and mitogenic effects on both normal and malignant cells (Tache et al., 1988). Two receptor subtypes have been well characterized, one having selectivity for GRP, the other having a greater selectivity for NMB (Kroog et al., 1995; Battey and Wada, 1991). Both subtypes have an architecture that resembles heptahelical G protein-coupled receptors (Kroog et al., 1995; Battey and Wada, 1991) and are coupled to similar signal transduction pathways: upon ligand binding, PLC activity ensues, resulting in protein kinase C activation and mobilization of intracellular calcium (Tache et al., 1988). Elevation of phospholipase D activity (Ben-Av et al., 1993; Hou et al., 1997) and tyrosine phosphorylation of intracellular proteins (Leeb-Lundberg and Song, 1991; Tsuda et al., 1997a) have also been described for these two receptor subtypes.

Recently, a 399-amino acid orphan receptor was identified in mammalian tissues (Gorbulev et al., 1992; Fathi et al., 1993) and has been proposed to represent a third mammalian Bn receptor subtype. This receptor, named bombesin receptor subtype 3 (BRS-3) because of its approximately 50% homology to GRP and NMB receptors (Fathi et al., 1993), has a pattern of expression that differs from the broader distribution described for the other established members of this receptor family. Studies of BRS-3 mRNA expression revealed a pattern limited to secondary spermatocytes (Fathi et al., 1993), pregnant uterus (Gorbulev et al., 1992), a few brain regions (Gorbulev et al., 1992) and tumor cell lines derived from human lung (Fathi et al., 1993), breast (Gorbulev et al., 1994) and epidermal tissues (Gorbulev et al., 1994). A recent study (Ohki-Hamazaki et al., 1997) using targeted disruption of the BRS-3 receptor demonstrates that it is important in regulating obesity and metabolic control of insulin and glucose. At present, the ligand is unknown, and there is a lack of cell lines expressing sufficient endogenous BRS-3 for study. However, recent studies using the newly discovered synthetic peptide agonist [DPhe⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6-14) in BALB 3T3 cells and NCI-H1299 lung cancer cells stably transfected with human BRS-3 suggest that BRS-3 employs signal transduction processes similar to those observed with the other Bn receptor subtypes (Mantey et al., 1997; Ryan et al., 1998).

In this study, we examined the ability of the novel peptide [DPhe⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6-14) to bind and stimulate intracellular signaling events in two lung cancer cell lines, NCI-N417 and NCI-H720, that natively express hBRS-3 (Fathi et al., 1993). In addition, we wanted to determine whether activation of native hBRS-3 receptors stimulated cell growth. With this compound, we demonstrate for the first time that in cells natively expressing this protein, BRS-3 receptors couple to phospholipase C to elicit IP metabolism and calcium mobilization as well as to phospholipase D to generate diacylglycerol. However, BRS-3 activation was not coupled to changes in activity of adenylate cyclase, nor did it cause cell proliferation. In addition, our results show that none of the currently known, naturally occurring Bn peptides were the putative ligand for hBRS-3. However, several synthetic peptides that function as GRP or NMB receptor antagonists also behaved as hBRS-3 antagonists, which could prove useful in determining the biological role of this receptor.

Finally, we examined the effect of [DPhe⁶,Ala¹¹,Phe¹³, Nle¹⁴]Bn(6-14) in a novel bioassay (McConnell et al., 1992) that permits real-time measurement of hBRS-3-mediated changes in metabolic rate in NCI-N417 cells. The discovery of cells that natively express functional hBRS-3 receptors and the discovery of the utility of metabolic rate activation as a bioassay represent important developments in our effort to understand the function of BRS-3.

	Materials and Methods

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Materials. The following were kindly provided by or obtained from the sources indicated: NCI-N417 human small cell lung carcinoma cells and NCI-H720 human non-small cell lung carcinoma cells (Herb Oie of the NCI-Navy Medical Oncology Branch, Naval Medical Center, Bethesda, MD), A375-6 human melanoma cells (Pius Hildebrand, University Hospital, Basel, Switzerland), RPMI 1640, DMEM, PBS, G418 sulfate and FBS (Gibco BRL, Grand Island, NY), Tris HCl (Bethesda Research Labs, Gaithersburg, MD), formic acid, ammonium formate, disodium tetraborate, IBMX, epinephrine, EDTA, -aminoethyl ether EGTA and soybean trypsin inhibitor (Sigma, St. Louis, MO), BSA fraction V (ICN Biomedicals Inc., Aurora, OH), aprotinin and HEPES (Boehringer Mannheim Biochemicals, Indianapolis, IN), AG 1-X8 resin (BIO-RAD, Richmond, CA), monobasic sodium phosphate (Mallinckrodt Inc., Paris, KY), Na[¹²⁵I] (2200 Ci/mmol), [2-³H]adenine (22 Ci/mmol), [methyl-³H]-thymidine (25 Ci/mmol) and [9,10(n)-³H]palmitic acid (53 Ci/mmol) (Amersham Life Science Inc., Arlington Heights, IL), [-³²P]ATP (3000 Ci/mmol) and myo-[2-³H] inositol (20 Ci/mmol) (Dupont/NEN, Boston, MA), 1,2,4,6-tetrachloro-3-6-diphenylglycouril (Iodo-Gen) (Pierce Chemical Co., Rockford, IL), silica gel G TLC plates (LK6D) (Whatman, Clifton, NJ), phosphatidylethanol (Avanti Polar Lipids, Birmingham, AL), PACAP-38, PACAP-27, Bn, neuromedin B, GRP, litorin, phyllolitorin, rohdei-litorin and ranatensin (Bachem, Torrence, CA), [DArg¹,DTrp^7,9,Leu¹¹]substance P and [DPro⁴,DTrp^7,9,10] substance P(4-11) (Peninsula Laboratories, Belmont, CA) and [Arg⁸] vasopressin (Novabiochem Corp., La Jolla, CA). [Phe¹³]bombesin, [Ser¹⁹]GRP(18-27) (frog GRP-10) and SAP-Bn were gifts from John Taylor of Biomeasure, Inc., Milford, MA. All other chemicals were reagent grade.

	Materials and Methods

Cell culture. NCI-N417, NCI-H720 and A375-6 cells were grown in RPMI-1640. Untransfected BALB 3T3 cells and BALB 3T3 cells transfected with human NMB receptors (Ryan et al., 1996) or human BRS-3 receptors (Mantey et al., 1997) were grown in DMEM. Both cell media were supplemented with 10% (v/v) FBS (plus 300 µg/ml G418 sulfate for the BALB 3T3 transfectants). All cell lines were incubated at 37°C in a 5% CO₂ atmosphere.

Isolation of RNA. Total RNA from all cell lines studied was isolated using the RNeasy Midi Kit (Qiagen, Inc., Chatsworth, CA) according to the instructions supplied by the manufacturer.

RT-PCR and Southern blotting. For RT-PCR, first strand cDNA was created using 1.0 µg of total cellular RNA with the First Strand Synthesis Kit (BRL/Gibco, Grand Island, NY). Gene-specific primers for hBRS-3 receptor (Mantey et al., 1997), hGRP receptor (Mantey et al., 1997) and hNMB receptor (Mantey et al., 1997) were used for amplification of first strand cDNA. To ensure that only cDNA could be used as a template, the primers were positioned on either side of an intron. PCR was performed using the GeneAmp PCR System 9600 thermal cycler (Perkin Elmer Cetus, Emeryville, CA) under routine conditions recommended by the manufacturer. Separation of PCR products was achieved by electrophoresis on 1.2% (w/v) SeaKem GTG agarose gels (FMC BioProducts, Rockland, ME). The products were then transferred to nitrocellulose filters. Hybridization was carried out at room temperature for 16 hr in a hybridization buffer containing 40% (v/v) formamide (Fluka Chemical, Switzerland), 4 × SSC (300 mM sodium chloride, 30 mM sodium citrate; Research Genetics, Huntsville, AL), 20 mM Tris (pH 7.5) (Quality Biological, Gaithersburg, MD), 10% (v/v) dextran sulfate (Oncor, Gaithersburg, MD), 1 × Denhardt solution (Digene Diagnostics, Beltsville, MD), 20 µg/ml sonicated herring sperm DNA (Digene Diagnostics, Beltsville, MD) and hGRP receptor, hNMB receptor or hBRS-3 receptor synthetic oligonucleotide probes end-labeled with [-³²P]ATP. The oligonucleotide probes contained gene-specific sequences between the gene-specific PCR primer pairs for each receptor. The nitrocellulose filters were washed with increasing stringency, with a final wash in 0.1 × SSC, 0.1% (v/v) at 25°C. After air-drying, the filters were exposed to XAR X-ray film (Kodak, Rochester, NY).

Preparation of peptides. The peptides were synthesized by solid-phase methods as previously described (Coy et al., 1988; Wang et al., 1990; Orbuch et al., 1993). Introduction of the reduced peptide bond () in various peptides was performed on methylbenzhydrylamine resin (Advanced Chem Tech, Louisville, KY) (Coy et al., 1988). DNal,Cys,Tyr,DTrp,Lys,Val,Cys,NalNH₂ was synthesized as described previously (Orbuch et al., 1993), using methylbenzhydrylamine resin. Various alkylamide and ester analogs of Bn(6-13) were synthesized in a standard Leu-O-polystyrene resin, using tosyl group protection for the imidazole group of His (Wang et al., 1990). Free peptide was removed from the resin after synthesis by transesterification with 10% triethylamine/methanol at 40°C for 48 hr. The peptides were first purified on a Sephadex G-25 column (2.5 × 90 cm), followed by preparative HPLC on a Vydac C₁₈ column (1.5 × 50 cm, bore size 10-15 µm). After rechromatography to achieve 97% purity, the peptides were characterized by amino acid analysis and matrix-assisted laser desorption mass spectroscopy (Finnegan, Hemel Hemstead, UK).

Preparation of ¹²⁵I-[DTyr⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6-14). ¹²⁵I-[DTyr⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6-14), with a specific activity of 2200 Ci/mmol, was prepared by methods described previously (Mantey et al., 1997). Briefly, 0.8 µg of an Iodo-Gen solution (0.01 µg in chloroform) was dried under nitrogen and washed with 100 µl of monobasic potassium phosphate (pH 7.4). To this solution, 20 µl of monobasic potassium phosphate (pH 7.4), 8 µg of [DTyr⁶,Ala¹¹, Phe¹³,Nle¹⁴]Bn(6-14) in 4 µl of water and 2 mCi (20 µl) of Na[¹²⁵I] were added, and the reaction was allowed to run at room temperature for 6 min after gentle mixing. The reaction was stopped by incubation of the mixture at 80°C for 60 min. The reaction mixture was added to a Sep-Pak (Waters Associates, Milford, MA), and free ¹²⁵I was eluted with 5 ml of water followed by 0.1% (v/v) trifluoroacetic acid (TFA). Radiolabeled peptide was removed by sequential elution (10 × 200 µl) with 60% acetonitrile in 0.1% TFA. The fractions with the highest radioactivity were pooled and purified by reverse-phase HPLC as previously reported (Mantey et al., 1997). Fractions that tested positive for radioactivity and binding were neutralized with 0.2 M Tris (pH 9.5) and stored with 0.5% BSA (w/v) at 20°C.

Binding of ¹²⁵I-[DTyr⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6-14) to NCI-N417 cells. NCI-N417 cells (1 × 10⁷ cells/ml) were incubated with 75 pM ¹²⁵I-labeled ligand for the indicated durations and temperatures in a binding buffer solution containing 24.5 mM HEPES (pH 7.4), 98 mM sodium chloride, 6 mM potassium chloride, 2.5 mM monobasic sodium phosphate, 5 mM sodium pyruvate, 5 mM sodium fumarate, 5 mM sodium glutamate, 2 mM glutamine, 11.5 mM glucose, 0.5 mM calcium chloride, 1.15 mM magnesium chloride, 0.01% soybean trypsin inhibitor, 0.2% (v/v) amino acid mixture, 0.2% (w/v) BSA and 0.1% (w/v) bacitracin. Nonsaturable binding was the amount of radioactivity seen with 75 pM ¹²⁵I-[DTyr⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6-14) in the presence of 1 µM [DTyr⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6-14), and was <10% of total binding in all experiments. Receptor affinities of ligands were determined using a least-squares curve-fitting program (LIGAND) and the Cheng-Prusoff equation.

Dissociation of ¹²⁵I-[DTyr⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6-14) from NCI-N417 cells. The time- and temperature-dependence of dissociation of ¹²⁵I-[DTyr⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6-14) from NCI-N417 cells was determined by incubation of the radioligand with the cells for 45 min at 25°C. The incubation mixture was then diluted 100-fold with binding buffer at the times indicated before filtering the cells on GF/B filters, which were washed and counted for saturably bound radioactivity.

Measurement of IP. NCI-N417 or NCI-H720 cells (5 × 10⁵ cells/ml) were subcultured into 75-cm² tissue culture flasks containing RPMI-1640 supplemented with 3 µCi/ml myo-[2-³H] inositol and 2% (v/v) FBS. After a 24-hr incubation period (37°C), the cells were washed and incubated for 10 min at 37°C with an equivalent volume of PBS (pH 7.0) containing 20 mM lithium chloride. The cells were then resuspended in an equivalent volume of IP assay buffer [135 mM sodium chloride, 20 mM HEPES (pH 7.4), 2 mM calcium chloride, 1.2 mM magnesium sulfate, 1 mM EGTA, 20 mM lithium chloride, 11.1 mM glucose and 0.05% BSA (w/v)], and 500 µl of cell suspension was added to tubes containing the peptides studied. For the hBRS-3-transfected BALB 3T3 cells, loading of myo-[2-³H] inositol and the assay protocol were as previously described (Benya et al., 1994). Briefly, cells were subcultured into 24-well plates (5 × 10⁴ cells/well) in their respective propagation media and then incubated at 37°C for 24 hr. The cells were incubated with 3 µCi/ml of myo-[2-³H] inositol in growth medium supplemented with 2% FBS for an additional 24 hr. Before assay, the 24-well plates were washed and incubated for 10 min at 37°C with 1 ml/well PBS (pH 7.0) containing 20 mM lithium chloride. The wash buffer was aspirated and replaced with 500 µl of assay buffer/well with or without any of the peptides studied. The experiments were terminated with 1 ml of ice-cold hydrochloric acid/methanol (0.1% v/v). After a 30-min extraction period (4°C), the samples were applied to glass columns containing 500 µl of a 1:3 (v/v) slurry of Dowex AG1-X8 anion exchange resin/distilled water to separate the various isomers. Total [³H]IP was isolated by a variation of a method described previously (Benya et al., 1994). Briefly, samples were loaded onto columns, washed with 5 ml of distilled water to remove [³H]inositol, and then washed with 2 ml of 5 mM disodium tetraborate/60 mM sodium formate solution to remove [³H]glycerophosphorylinositol. The columns were then eluted with 2 ml of 1 mM ammonium formate/100 mM formic acid solution to elute total [³H]IP. Each of the eluates was collected and mixed with 10 ml of Hydrofluor scintillation cocktail (National Diagnostics, Atlanta, GA), and the radioactivity was measured in a scintillation counter.

[Ca⁺⁺]_i. Cells harvested by centrifugation (2 min, 300 × g) were resuspended in an assay buffer [24.5 mM HEPES (pH 7.4), 98 mM sodium chloride, 6 mM potassium chloride, 2.5 mM monobasic sodium phosphate, 5 mM sodium pyruvate, 5 mM sodium fumarate, 5 mM sodium glutamate, 2 mM glutamine, 11.5 mM glucose, 1.45 mM calcium chloride, 1.15 mM magnesium chloride, 0.01% soybean trypsin inhibitor, 0.2% (v/v) amino acid mixture, and 0.2% BSA (w/v)] to a concentration of 1.5 × 10⁶ cells/ml and incubated with 2.5 µM Fura-2/AM (Molecular Probes, Eugene, OR) for 30 min at 37°C followed by 15 min at 25°C. After two washes with assay buffer, 2 ml of cell suspension were placed in a Delta PTI Scan 1 spectrofluorimeter (Photon Technology International, South Brunswick, NJ) equipped with a stir bar and water bath (37°C). Fluorescence was measured at dual excitation wavelengths of 340 nm and 380 nm, using an emission wavelength of 510 nm. Autofluorescence was corrected for by running a sample of unlabeled cells in identical experimental conditions.

PLD assay. PLD activity was determined using a modification of a method previously reported (Cook et al., 1991). NCI-N417 cells (5 × 10⁶ cells/ml) were incubated in RPMI-1640 containing 2% FBS (v/v) for 24 hr (37°C) before the experiments. The cells were then labeled with 4 µCi/ml [³H]palmitic acid in 2% serum-supplemented media for 24 hr at 37°C. After this period, the cells were washed and preincubated in PLD buffer [serum-free RPMI-1640, 20 mM HEPES (pH 7.4), and 1% BSA (w/v)] for 30 min (37°C) and then incubated for an additional 5 min in fresh PLD buffer containing 1% (v/v) ethanol. To start the assay, the cells were incubated in fresh PLD buffer containing [DPhe⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6-14), GRP or NMB at the indicated concentrations with 1% (v/v) ethanol for 30 min. The 30 min assay period was used because previously performed time-course experiments showed that this was the interval needed for measuring maximal PLD activity (data not shown). The experiments were terminated by the addition of 1.4 ml of methanol after removal of medium. After extraction with an equivalent volume of chloroform (15 min, 25°C), the samples were mixed with 585 µl of water and centrifuged (2500 × g, 5 min) to separate the phases. The organic phase was collected and dried under nitrogen gas and then was redissolved in 30 µl of chloroform/methanol (19:1, v/v). Before thin-layer chromatography (TLC) on Whatman TLC plates, PETH standard was added to each sample. Lipids were separated using a solvent system containing 2,2,4-trimethylpentane/ethyl acetate/acetic acid/water (5:12:2:10, by volume). Upon staining with iodine vapor, [³H]PETH was identified as the band co-migrating with the PETH standard. The bands were scraped into scintillation vials and mixed with Hydrofluor scintillation cocktail, and the radioactivity was measured in a scintillation counter.

Microphysiometry. The effect of various natural and synthetic Bn-related peptides on the metabolic activity of NCI-N417 cells was examined using the Cytosensor Microphysiometer system (Molecular Devices, Sunnyvale, CA), which employs a light-addressable potentiometric sensor to detect pH changes in the extracellular fluid (McConnell et al., 1992). Briefly, NCI-N417 cells were harvested by centrifugation and resuspended to a concentration of 2 × 10⁷ cells/ml in assay medium [bicarbonate-free DMEM (pH 7.4) supplemented with 44 mM sodium chloride and 0.1% (w/v) BSA]. The cell solution was mixed 1:1 with Agarose Cell Entrapment Medium (Molecular Devices, Sunnyvale, CA), and 10 µl aliquots of this solution were seeded into 12-mm capsule cups and placed into the Cytosensor. The assembly was equilibrated in assay medium for 1 hr at a perfusion rate of 100 µl/min. The cells were exposed to the various peptides for 4 min, and the acidification rates were determined during the last 30 sec of the peptide exposure interval. A temperature of 37°C was maintained throughout the equilibration and experimental periods.

cAMP. NCI-N417 cells (2 × 10⁶ cells/ml) were incubated with RPMI-1640 medium supplemented with 2% FBS (v/v) and 2 µCi/ml [³H]adenine for 24 hr at 37°C. The cells were harvested by centrifugation and resuspended into an equivalent volume of RPMI-1640 containing 1% BSA (w/v) and 0.5 mM IBMX. Then 500 µl aliquots of cell suspension were added to tubes containing the indicated agents at the indicated concentrations and incubated for 30 min at 37°C. Reactions were terminated by the addition of 100 µl of stopping solution [2% SDS (v/v), 5 mM cAMP] followed by 900 µl of ice-cold Tris (50 mM, pH 7.4). Samples were stored at 20°C until analyzed.

[³H]-Thymidine incorporation. The ability of hBRS-3 activation to stimulate DNA synthesis was examined using a modification of a previously described [³H]-thymidine incorporation assay (Benya et al., 1994). Briefly, 100-µl of 2 × 10⁴ NCI-N417 cells/well in serum-free RPMI-1640 medium were plated into 96-well plates. After a 24-hr incubation at 37°C, 1 µCi/well of [methyl-³H]-thymidine was added with 100 µl of serum-free RPMI-1640 medium containing no peptide, 30 nM or 1000 nM [DPhe⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6-14), or medium containing 10% FBS (v/v). After incubation for an additional 24 hr at 37°C, the radiolabeled DNA was collected on glass-fiber filters (Wallac, Gaithersburg, MD) using a cell harvester (Tomtec, Orange, CT), and the radioactivity was measured in a scintillation counter.

Cell proliferation. The ability of hBRS-3 activation to stimulate cell proliferation was determined using the CellTiter 96 AQ_ueous cell proliferation assay kit (Promega, Madison, WI). The method, which is a modification of the MTT assay (Carmichael et al., 1988), employs the yellow tetrazolium dye MTS and the electron-coupling reagent phenazine ethosulfate. The MTS compound is reduced by viable cells to purple, water-soluble formazan product and is a colorimetric index of cell proliferation. NCI-N417 cells (5 × 10³/well) were plated in RPMI-1640 medium containing 2% FBS (v/v) and incubated for 24 hr at 37°C. In contrast to the [³H]-thymidine assay, 2% FBS (v/v) was included in all samples because there was a significant loss in cell viability after 3 days in the absence of FBS. After addition of medium containing no peptide, 30 nM or 1000 nM [DPhe⁶,Ala¹¹,Phe¹³, Nle¹⁴]Bn(6-14), or 10% FBS, the cells were allowed to incubate at 37°C. On the indicated days, 20 µl of MTS solution was added, and the plates were incubated in the dark for 3 hr at 37°C. The absorbance at 490 nM was obtained using a spectrophotometric plate reader (Molecular Devices Corp., Sunnyvale, CA).

Statistical analysis. Data plotting and iterative curve fitting were performed with KaleidaGraph graphing software (Synergy Software, Reading, PA). Analysis of Schild plots and statistical analysis of the data were performed using Statview version 1.01 (BrainPower, Inc., Calabasas, CA). Student's t test was used to determine the statistical significance of the difference between group means. P values of less than .05 were considered significant.

	Results

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NCI-N417 and NCI-720 cells have been reported to have detectable levels of hBRS-3 mRNA (Fathi et al., 1993). To determine whether these cell lines expressed hBRS-3 receptor or any other Bn receptor, we used RT-PCR and Southern blot analysis (fig. 1). NCI-N417 cells expressed only hBRS-3 receptors, whereas NCI-H720 cells expressed both hBRS-3 and hGRP receptors. Neither cell line expressed hNMB receptors. To determine whether these receptors were functional, we examined the ability of [DPhe⁶,Ala¹¹,Phe¹³, Nle¹⁴]Bn(6-14), GRP and NMB to stimulate an increase in [³H]IP in both cell lines (table 1). In the NCI-N417 cells, only [DPhe⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6-14) was capable of stimulating a significant release of [³H]IP at both 10 nM and 1 µM concentrations. [DPhe⁶]Bn(6-13) methyl ester, a GRP receptor-specific antagonist that has low affinity for BRS-3 and NMB receptors (Mantey et al., 1997) did not inhibit this increase. Neither GRP nor NMB had an agonist effect at 1 µM. In the NCI-H720 cells, both [DPhe⁶,Ala¹¹,Phe¹³, Nle¹⁴]Bn(6-14) and GRP stimulated an elevation of [³H]IP at 10 nM and 1 µM concentrations, and agonist activity was observed with 1 µM NMB (table 1). [DPhe⁶]Bn(6-13) methyl ester blocked the effect of GRP and NMB, attenuated the rise in [³H]IP seen with 10 nM [DPhe⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6-14) by 18% and had a smaller but statistically significant antagonist effect against 1 µM [DPhe⁶,Ala¹¹,Phe¹³,Nle¹⁴] Bn(6-14) (11%). The RT-PCR and [³H]IP data suggested that the NCI-H720 cells contained hGRP receptors and that these were present in sufficient numbers to result in GRP-stimulated increases in [³H]IP, so we used only the NCI-N417 cells for assessing hBRS-3 activation in the remaining experiments, because they possessed only hBRS-3 receptors.

View larger version (34K): [in this window] [in a new window]   Fig. 1.   Autoradiograph of Southern blot after RT-PCR using gene-specific primers for hBRS receptors, hGRP receptors and hNMB receptors in NCI-N417 cells, NCI-H720 cells and other cells known to contain these receptors. Reverse transcriptase was performed using total cellular RNA from each cell line as described in "Materials and Methods." PCR was performed with gene-specific primers for hBRS-3 receptors, hGRP receptors and hNMB receptors. Identification of Bn receptor subtypes was carried out with 32P-radiolabeled, gene-specific probes as described in "Materials and Methods." hBRS-3-transfected BALB 3T3 cells served as the positive control for hBRS-3 receptors, A375-6 cells for hGRP receptors and hNMB receptor-transfected BALB 3T3 cells for hNMB receptors. dH2O represents a PCR reaction where deionized water was substituted for template DNA. The top panel shows the results using a hBRS-3 receptor-specific probe, the middle panel the results using a hGRP receptor-specific probe and the bottom panel the results using a hNMB receptor-specific probe. This experiment is representative of two others.

View this table: [in this window] [in a new window]   TABLE 1 Ability of [DPhe6,Ala11,Phe13,Nle14]Bn(6-14), GRP and NMB to alter [3H]IP in NCI-N417 and NCI-H720 cells in the presence or absence of a hGRP receptor antagonist NCI-N417 and NCI-H720 cells were incubated with GRP, NMB or [DPhe6,Ala11,Phe13,Nle14]Bn(6-14) at the indicated concentrations for 45 min in the presence or absence of the hGRP receptor antagonist [DPhe6]Bn(6-13)methyl ester (ME). Results are expressed as the ratio of total [3H]IP released in the presence of peptide (Exp) to that released in the absence of peptide (Con). Each value represents the means ± S.E. of at least four experiments performed in duplicate. The control values in NCI-N417 and NCI-H720 cells were 510 ± 72 and 304 ± 26 dpm, respectively. The values with 1 µM [DPhe6,Ala11,Phe13,Nle14]Bn(6-14) were 1187 ± 193 and 586 ± 64 dpm for the NCI-N417 and NCI-H720 cells, respectively.

We examined the ability of ¹²⁵I-[DTyr⁶,Ala¹¹,Phe¹³, Nle¹⁴]Bn(6-14), which binds to hBRS-3 receptors (Mantey et al., 1997), to bind to NCI-N417 cells. Binding was time- and temperature-dependent (fig. 2), reaching a maximum by 20 min at 37°C and 30 min at 22°C, and remained constant for 40 and 30 min, respectively. At both temperatures, the binding was markedly attenuated (>90%) by the addition of 1 µM [DTyr⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6-14). At an incubation temperature of 4°C, saturable binding was reduced to 12% to 14% of the maximal binding seen at 37°C and 22°C. The rate of dissociation was temperature-dependent; as shown in figure 3, 30% of the ligand dissociated within 10 min, and an additional 30% dissociated over the next 50 min at 37°C, but the rate of dissociation was slowed sufficiently at 4°C so that only 10% dissociated by 60 min.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Time- and temperature-dependence of 125I-[DTyr6,Ala11,Phe13,Nle14]Bn(6-14) binding to NCI-N417 cells. NCIN417 cells (1 × 107 cells/ml) were incubated with 75 pM 125I-[DTyr6,Ala11,Phe13,Nle14]Bn(6-14) at the indicated temperatures. At each specified time, 100-µl aliquots were removed. Total binding and nonsaturable binding (binding in the presence of 1 µM [DTyr6,Ala11,Phe13,Nle14]Bn(6-14) were determined at each temperature by means of centrifugation as described in "Materials and Methods." Results are the means ± S.E. from at least three experiments performed in duplicate.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Time- and temperature-dependence of dissociation of 125I-[DTyr6,Ala11,Phe13,Nle14]Bn(6-14) from NCI-N417 cells. After incubation of NCI-N417 cells (1.5 × 107) for 45 min at 25°C with 75 pM 125I-[DTyr6,Ala11,Phe13,Nle14]Bn(6-14), the cells were diluted 100-fold in incubation buffer at 37°C or 4°C and incubated for the indicated time before filtration on GF/B filters. Results are expressed as the percentage of saturably bound ligand at time 0 (percent initial) and are the means ± S.E. from at least three experiments performed in duplicate.

[DTyr⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6-14) attenuated binding of ¹²⁵I-[DTyr⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6-14) in a concentration-dependent manner in the NCI-N417 cells (fig. 4). Detectable inhibition was observed at 0.1 nM [DTyr⁶,Ala¹¹,Phe¹³, Nle¹⁴]Bn(6-14), half-maximal inhibition at 7.4 nM and complete inhibition at 1 µM. Analysis of the [DTyr⁶,Ala¹¹,Phe¹³, Nle¹⁴]Bn(6-14) inhibition curve (fig. 4, insert) demonstrated that the binding was best fitted with a single-site model, using least-squares curve-fitting analysis (LIGAND). The affinity of [DTyr⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6-14) for the hBRS-3 receptor on NCI-N417 cells was 7.4 ± 1.5 nM, with a binding capacity of 1.1 ± 0.2 fmol/mg protein (68 ± 10 fmol/10⁶ cells). The NCI-N417 cells had little or no affinity for Bn; 3 µM did not cause a significant decrease in binding of ¹²⁵I-[DTyr⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6-14) (fig. 5). GRP caused detectable binding at 3 µM, and NMB at 1 µM (fig. 5), which showed that the hBRS-3 receptor had a very low affinity (>5000 nM) for each of these naturally occurring mammalian Bn peptides.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Receptor number and affinity of 125I-[DTyr6,Ala11,Phe13,Nle14]Bn(6-14) for BRS-3 receptors on NCI-N417 cells. NCI-N417 cells (1 × 107 cells/ml) were incubated for 45 min at 25°C with 75 pM 125I-[DTyr6,Ala11,Phe13, Nle14]Bn(6-14) with or without [DTyr6,Ala11,Phe13,Nle14]Bn(6-14) at the concentrations indicated. Results are expressed as the percentage of saturable binding seen without the addition of [DTyr6,Ala11,Phe13,Nle14]Bn(6-14). The insert shows the dose-inhibition data plotted in the form of Scatchard. Results are means ± S.E. from at least three experiments using duplicate determinations.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Comparison of the ability of [DTyr6,Ala11,Phe13,Nle14]Bn(6-14) and Bn, GRP or NMB to inhibit binding of 125I-[DTyr6,Ala11,Phe13, Nle14]Bn(6-14) to NCI-N417 cells. The experimental conditions were the same as outlined in figure 4, except that the indicated concentrations of [DTyr6,Ala11,Phe13,Nle14]Bn(6-14), Bn, GRP or NMB were added. Results are expressed as the percentage of saturable binding without unlabeled peptide (percent control). Results are the means ± S.E. from at least three experiments, and in each experiment the data-points were determined in duplicate.

To determine whether any of the known naturally occurring Bn-related peptides interacted with native hBRS-3 receptors, we determined the affinities of 11 other natural occurring peptides of the bombesin family for the hBRS-3 receptor in NCI-N417 cells (table 2). None of the 11 peptides had high affinity for the hBRS-3 receptor on NCI-N417 cells, and none had an affinity greater than 3 µM. Of the 11 evaluated, ranatensin and NMB had the highest affinity for hBRS-3 receptors, which was >3 µM for both peptides (table 2). Similar results were obtained previously in hBRS-3-transfected BALB 3T3 and NCI-H1299 cells (Ryan et al., 1997), and none of the natural peptides had high affinity for hBRS-3 receptors (table 2).

View this table: [in this window] [in a new window]   TABLE 2 Affinity of various naturally occurring Bn-related peptides, synthetic GRP receptor (GRP-R) agonists and synthetic Bn receptor (BN-R) antagonists for NCI-N417 cells or cells transfected with hBRS-3 Cells (1-10 × 106/ml) were incubated with 75 pM 125I-[DTyr6,Ala11,Phe13,Nle14]Bn(6-14) for 45 min at 22°C. Increasing concentrations of unlabeled peptide were added, and the dose-inhibition curves were analyzed by a least-squares curve-fitting program (LIGAND). Ki values were calculated using the method of Cheng and Prusoff (Cheng and Prusoff, 1973) and are the means ± S.E. from at least three experiments performed in duplicate. a Binding data for hBRS-3 transfected cells are from (Mantey et al., 1997); >10,000 means the affinity was greater than 10,000 nM.

Numerous synthetic peptides, which behave as agonists or antagonists at GRP or NMB receptors, have been described (Jensen and Coy, 1991; Wang et al., 1990). Twenty-one of these compounds, which are representative of the different types of synthetic peptides described, were tested for their ability to interact with hBRS-3 in NCI-N417 or hBRS-3-transfected cells. Representative members of four classes of the Bn receptor antagonists (Jensen and Coy, 1991) had a much lower affinity (i.e., >4000 nM) for hBRS-3 receptors than reported for the hGRP or hNMB receptors, which included a DPhe¹²-substituted analog (analog 20); two Bn pseudopeptide GRP analogs (analogs 21 and 22); two DPro¹³ Bn pseudopeptides (analogs 23 and 24) and eight des-Met¹⁴ amides, esters or alkylamides (analogs 26-33) (Wang et al., 1990). Two classes of Bn receptor antagonists, the D-substituted substance P analogs (analogs 35 and 36), which are broad-spectrum neuropeptide receptor antagonists, and a somatostatin octapeptide analog (analog 37), had low affinity (4-9 µM) for the hBRS-3 receptor (table 2; fig. 6), which is similar to that reported for these antagonists for the hGRP or hNMB receptors. Three synthetic Bn-related agonists (analogs 16-18), with substitutions similar to [DPhe⁶,Ala¹¹,Phe¹³, Nle¹⁴]Bn(6-14) (analog 15), and a NMB analog (analog 19) also had low affinity for hBRS-3 receptors (table 2).

View larger version (34K): [in this window] [in a new window]   Fig. 6.   Ability of various Bn receptor antagonists to inhibit binding of 125I-[DTyr6,Ala11,Phe13,Nle14]Bn(6-14) to BRS-3 receptors in NCI-N417 cells. The experimental conditions were the same as described in the figure 4 legend, except that the indicated concentrations of antagonists were added. Results are expressed as the percentage of saturable binding in the absence of unlabeled peptide and represent the means ± S.E. from at least three experiments where each point was determined in duplicate.

However, five peptides (litorin, phyllolitorin, rohdei-litorin, alytesin and NMB) had affinities 5 µM in both transfectants (table 2). Five peptides (SAP-Bn, [Phe¹³]Bn, ranatensin, Xenopus NMB and [Leu⁸]phyllolitorin) had affinities >5 µM in the BALB 3T3 cells and three ([Phe¹³]Bn, ranatensin and Leu⁸]phyllolitorin) in the H1299 cells. Three peptides in the BALB 3T3 transfectants (Bn, GRP and frog GRP-10) and five in the H1299 transfectants (Bn, SAP-Bn, GRP, frog GRP-10 and Xenopus NMB) had almost no affinity for hBRS-3 receptors (table 2).

To determine whether any of the naturally occurring Bn-related peptides activated hBRS-3 receptors, we examined the ability of a number of these peptides to stimulate [³H]IP release on NCI-N417 cells and hBRS-3-transfected BALB 3T3 cells (table 3), because previous studies showed that transfected hBRS-3 receptors couple to phospholipase C (Ryan et al., 1998; Wu et al., 1996; Fathi et al., 1993; Mantey et al., 1997). None of the 10 naturally occurring Bn peptides that we studied, at a concentration of 1 µM, elicited a significant [³H]IP response in the NCI-N417 cells, whereas 1 µM [DPhe⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6-14), a synthetic Bn analog, stimulated a 2-fold increase in total [³H]IP (table 3). At higher concentrations (i.e., >1000 nM) NMB, but not GRP, stimulated a detectable response (fig. 7). In the hBRS-3-transfected BALB 3T3 cells, five naturally occurring Bn-related peptides (Bn, GRP, NMB, SAP-Bn and frog GRP-10) did not cause an increase in [³H]IP, whereas five naturally-occurring peptides (litorin, phyllolitorin, rohdei-litorin, [Phe¹³]Bn and ranatensin) did (table 3).

View larger version (23K): [in this window] [in a new window]   Fig. 7.   Effect of GRP, NMB and various synthetic Bn analogs on [3H]IP formation in NCI-N417 cells. NCI-N417 cells were incubated with GRP, NMB or the indicated synthetic analogs for 45 min at the specified concentrations. Values represent the percent of total [3H]IP release stimulated by 1 µM [DPhe6,Ala11,Phe13,Nle14]Bn(6-14) and are the means ± S.E. from at least three experiments performed in duplicate. The control and 1 µM [DPhe6,Ala11,Phe13,Nle14]Bn(6-14)-stimulated values were 560 ± 41 dpm and 1312 ± 135 dpm, respectively (n = 7).

[DPhe⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6-14) and three other peptides, Ac-NMB(3-10), [DPhe⁶]Bn(6-13) propylamide and [DPhe⁶,Phe¹³]Bn(6-13) propylamide, which have been reported to have high affinity for transfected hBRS-3 receptors (Wu et al., 1996; Ryan et al., 1998), were also studied for their ability to activate phospholipase C (table 3). [DPhe⁶,Ala¹¹, Phe¹³,Nle¹⁴]Bn(6-14) and two of the other peptides, Ac-NMB(3-10) and [DPhe⁶]Bn(6-13) propylamide, caused detectable stimulation of [³H]IP at concentrations of 1 µM in both NCI-N417 cells and hBRS-3-transfected BALB 3T3 cells (table 3). Dose-response curves for these peptides (fig. 7) demonstrated that each of these three peptides stimulated [³H]IP release in a concentration-dependent manner in the NCI-N417 cells with EC values of 25 ± 6 nM for [DPhe⁶,Ala¹¹, Phe¹³,Nle¹⁴]Bn(6-14), 1500 ± 140 nM for Ac-NMB(3-10) and 2760 ± 900 nM for [DPhe⁶]Bn(6-13) propylamide (fig. 7). In contrast, [DPhe⁶,Phe¹³]Bn(6-13) propylamide had no detectable agonist activity, even up to concentrations of 10 µM (table 3; fig. 7).

One member of each of the five classes of GRP or NMB receptor antagonists was examined for intrinsic agonist activity by altering phospholipase C activity through the hBRS-3 receptor (table 4, fig. 8). [DArg¹,DTrp^7,9,Leu¹¹] substance P stimulated a significant increase in [³H]IP in the NCI-H417 cells and hBRS-3-transfected BALB 3T3 cells at a concentration of 100 µM but had no agonist activity at lower concentrations (data not shown). Each of the other Bn receptor antagonists, [DPhe⁶]Bn(6-13) methyl ester, [(3-Ph-Pr⁶)-His⁷,DAla¹¹,DPro¹³,(13-14),Phe¹⁴]Bn(6-14)NH₂, [DPhe⁶,Leu¹³, (CH₂NH),Cpa¹⁴]Bn(6-14), DNal,Cys,Tyr,DTrp,Lys,Val,Cys,NalNH₂ and [DPro⁴,DTrp^7,9,10]SP(4-11), at concentrations up to 100 µM, had no agonist activity (data not shown).

View this table: [in this window] [in a new window]   TABLE 4 Ability of various GRP receptor and NMB receptor antagonists to inhibit [3H]IP generation in cells natively expressing hBRS-3 receptors or cells transfected with hBRS-3 receptors NCI cells or NCI-H1299 cells transfected with hBRS-3 receptors were incubated with 100 nM [DPhe6,Ala11,Phe13,Nle14]Bn(6-14) for 45 min with or without 100 µM of each of the listed peptides. The results are expressed as the percent of [3H]IP stimulated in the presence of antagonist compared with [DPhe6,Ala11,Phe13, Nle14]Bn(6-14) alone and are the means ± S.E. from at least three experiments. The [3H]IP values for control were 742 ± 65 dpm for the NCI-N417 cells and 1373 ± 225 dpm for the transfected cells. The values with 100 nM [DPhe6,Ala11, Phe13,Nle14]Bn(6-14) were 2718 ± 357 dpm for the NCI-N417 cells and 13472 ± 2716 dpm for the transfected cells.

View larger version (31K): [in this window] [in a new window]   Fig. 8.   Comparison of various Bn receptor antagonists to inhibit [3H]IP formation by [DPhe6,Ala11,Phe13,Nle14]Bn(6-14) in NCI-N417 cells. NCI-N417 cells were incubated with 100 nM [DPhe6,Ala11,Phe13,Nle14]Bn(6-14) with each of the indicated peptides at the indicated concentrations for 35 min. Values represent the percent increase in [3H]IP seen with 100 nM [DPhe6,Ala11,Phe13,Nle14]Bn(6-14). The results are the means ± S.E. from at least three experiments performed in duplicate. The control value was 742 ± 65 dpm, and the 100 nM [DPhe6,Ala11,Phe13,Nle14]Bn(6-14)-stimulated value was 1718 ± 357 dpm.

To determine the antagonist activities of each of the four Bn receptor antagonists that lacked agonist activity, we examined their ability to inhibit increases in [³H]IP caused by 100 nM [DPhe⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6-14) (table 4; fig. 8). The reduced peptide bond Bn analog [DPhe⁶,Leu¹³,(CH₂NH), Cpa¹⁴]Bn(6-14), the DPro¹³ Bn pseudopeptide [(3-Ph-Pr⁶)-His⁷,DAla¹¹,DPro¹³,(13-14),Phe¹⁴]Bn(6-14)NH₂, the somatostatin octapeptide analog DNal,Cys,Tyr,DTrp,Lys,Val,Cys,NalNH₂ and the D-amino acid substance P(4-11) analog [DPro⁴,DTrp^7,9,10]substance P(4-11) all significantly inhibited 100 nM [DPhe⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6-14)-stimulated [³H]IP (table 4). DNal,Cys,Tyr,DTrp,Lys,Val,Cys,NalNH₂, which is a NMB receptor-selective antagonist, was the most potent antagonist, causing detectable inhibition at 1 µM, half-maximal inhibition at 2 µM and 90% inhibition at 30 µM (fig. 8). [DPhe⁶]Bn(6-13) methyl ester was a weak inhibitor, attenuating the response by only 12% to 26% at the highest concentration tested (table 4).

Because previous studies with hBRS-3-transfected cells revealed that activation of hBRS-3 receptors caused cytosolic calcium release (Ryan et al., 1998), we evaluated the effect of [DPhe⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6-14) on calcium mobilization in the NCI-N417 cells. [DPhe⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6-14) (100 nM) stimulated a rapid rise in cytosolic calcium, which reached maximal levels in 13 sec and returning to basal levels in 1 min (fig. 9, left panel). Both GRP and NMB (1 µM) failed to stimulate calcium release (fig. 9, left panel). When EGTA was added to remove extracellular calcium, the magnitude of the calcium transient was reduced by 25%, the latency to reach peak levels was increased and the return to basal levels was faster than that seen with cells in calcium-containing buffer (fig. 9, right panel). Both the magnitude of released calcium and the time to reach the peak of the transient were concentration-dependent (fig. 10, left panel). [DPhe⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6-14) caused a detectable response at 1 nM and a maximal 3.6-fold increase at 1 µM. Analysis of the dose-response data by nonlinear, iterative curve fitting (fig. 10, right panel) revealed an EC₅₀ of 14 ± 7.1 nM.

View larger version (21K): [in this window] [in a new window]   Fig. 9.   Ability of [DPhe6,Ala11,Phe13,Nle14]Bn(6-14), NMB and GRP to stimulate Ca++ mobilization and the effect of extracellular Ca++ on [DPhe6,Ala11, Phe13,Nle14]Bn(6-14)-induced Ca++ release in NCI-N417 cells. NCI-N417 cells were loaded with Fura-2/AM and assayed under conditions outlined in "Materials and Methods." Left panel) Cells were treated with GRP, NMB or [DPhe6,Ala11,Phe13,Nle14]Bn(6-14) with the indicated concentrations at the indicated times. Right panel) Cells were stimulated with 100 nM [DPhe6,Ala11,Phe13,Nle14]Bn(6-14) in the presence or absence of 1.5 mM EGTA. Tracings are from a typical experiment performed three times.

View larger version (21K): [in this window] [in a new window]   Fig. 10.   Concentration-dependence of [DPhe6,Ala11,Phe13,Nle14]Bn(6-14) on Ca++ mobilization in NCI-N417 cells. Fura-2/AM-loaded cells were stimulated with [DPhe6,Ala11,Phe13,Nle14]Bn(6-14) at the concentrations indicated. Each point represents the change in calcium from basal levels to the peak of each transient observed with the indicated concentrations of [DPhe6,Ala11,Phe13,Nle14]Bn(6-14), and each is displayed as a percent of the change seen with 1 µM [DPhe6,Ala11,Phe13,Nle14]Bn(6-14). The control and stimulated values were 45 ± 2 nM and 162 ± 5 nM, respectively, and are the means ± S.E. from five experiments. Insert) While monitoring fluorescence, we stimulated Fura-2/AM-loaded NCI-N417 cells with [DPhe6,Ala11,Phe13,Nle14]Bn(6-14) at the concentrations indicated. The figure is from a representative experiment performed at least three times.

To determine whether hBRS-3 receptor activation affected the metabolic state of NCI-N417 cells, we examined the ability of GRP, NMB and [DPhe⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6-14) to stimulate extracellular acidification (fig. 11). [DPhe⁶,Ala¹¹, Phe¹³,Nle¹⁴]Bn(6-14) stimulated a 11 ± 0.8% increase in the acidification rate, which returned to basal levels in 6 to 8 min. The cells could be repeatedly stimulated, and the magnitude of the response from successive, equivalent doses of [DPhe⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6-14) was not significantly different from the initial treatment (data not shown). Neither GRP nor NMB was able to elicit acidification, and the GRP receptor antagonist [DPhe⁶]Bn(6-13) methyl ester was ineffective at attenuating the stimulation of acidification by [DPhe⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6-14) (fig. 11). When examined in more detail, the response seen with the synthetic peptide [DPhe⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6-14) was shown to be concentration-dependent, having an EC₅₀ of 4.3 ± 1.6 nM (fig. 12).

View larger version (19K): [in this window] [in a new window]   Fig. 11.   Ability of NMB, GRP and [DPhe6,Ala11,Phe13,Nle14]Bn(6-14) to induce metabolic changes in NCI-N417 cells. Cells were prepared as described in "Materials and Methods." Cells were treated with 100 nM NMB, GRP or [DPhe6,Ala11,Phe13,Nle14]Bn(6-14), and one sample was pretreated with 100 nM [DPhe6]Bn(6-14) methyl ester (ME) before the addition of [DPhe6,Ala11,Phe13,Nle14]Bn(6-14). The values represent the rate of acidification compared with the unstimulated basal rate and are representative of three independent experiments.

View larger version (19K): [in this window] [in a new window]   Fig. 12.   Ability of [DPhe6,Ala11,Phe13,Nle14]Bn(6-14) to stimulate metabolic responses in NCI-N417 cells. Cells were prepared as described in "Materials and Methods." The acidification rate was monitored during addition of [DPhe6,Ala11,Phe13,Nle14]Bn(6-14) at the concentrations indicated. Values are the rate of acidification expressed as a percent of the rate seen with 100 nM [DPhe6,Ala11,Phe13,Nle14]Bn(6-14). The control rate was 154 ± 15 µV/sec, and the maximal stimulated rate was 178 ± 18 µV/sec. The insert shows the data from a typical dose-response experiment, which was performed at least three separate times.

Because the hBRS-3 structurally related receptors, the mammalian GRP and the NMB receptor, have been shown to couple to phospholipase D and promote diacylglycerol formation (Pettitt and Wakelam, 1993; Hou et al., 1997), we examined the effect of NMB, GRP and [DPhe⁶,Ala¹¹,Phe¹³, Nle¹⁴]Bn(6-14) on phospholipase D activity in NCI-N417 cells using the transphosphatidylation assay. Neuromedin B and GRP, at a concentration of 100 nM, did not cause a significant increase in phospholipase D activity (fig. 13). However, [DPhe⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6-14) stimulated a significant increase in phospholipase D activity in NCI-N417 cells at 10 nM and 100 nM; increases of 105 ± 36% and 157 ± 47%, respectively, were observed.

View larger version (15K): [in this window] [in a new window]   Fig. 13.   Ability of [DPhe6,Ala11,Phe13,Nle14]Bn(6-14), GRP and NMB to stimulate PLD activity in NCI-N417 cells. Formation of [3H]PETH was measured in NCI-N417 cells prelabeled with [3H]palmitate for 24 hr during a 30-min incubation at 37°C with or without the indicated concentrations of [DPhe6,Ala11,Phe13,Nle14]Bn(6-14), GRP or NMB. Results are expressed as the percent increase over basal (control) and are the means ± S.E. from three experiments performed in triplicate. The control and maximal stimulated (100 nM) [DPhe6,Ala11,Phe13,Nle14]Bn(6-14) values were 5336 ± 834 dpm and 10396 ± 774 dpm, respectively. * Significantly greater than control (P < .05). ** Significantly greater than control (P < .01).

Because it had previously been shown that natively expressed GRP receptors in Swiss 3T3 fibroblasts could stimulate cAMP release upon receptor activation (Millar and Rozengurt, 1988), we studied the ability of [DPhe⁶,Ala¹¹,Phe¹³, Nle¹⁴]Bn(6-14) and various agonists known to activate adenylate cyclase via receptor activation. As shown in table 5, no stimulatory effect was observed with [DPhe⁶,Ala¹¹,Phe¹³, Nle¹⁴]Bn(6-14), vasopressin or epinephrine. Only two agents, PACAP-27 and PACAP-38, were capable of stimulating a significant increase in cAMP similar to that seen with forskolin, a direct activator of adenylate cyclase (table 5).

View this table: [in this window] [in a new window]   TABLE 5 Comparison of the ability of [DPhe6,Ala11,Phe13,Nle14]Bn(6-14) and other agents to elevate cAMP levels in NCI-N417 cells NCI-N417 cells were incubated with each of the indicated agents at the indicated concentrations for 30 min. Results are expressed as the ratio of total [3H]cAMP released in the presence of agonists (Exp) to that released in the absence of agonists (Con). Each value represents the means ± S.E. of at least three experiments performed in duplicate. The control value was 85 ± 11 cpm, and the maximally stimulated value was 332 ± 54 cpm.

To determine whether hBRS-3 receptor activation resulted in DNA synthesis and proliferation, we examined the ability of [DPhe⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6-14) to stimulate an increase in [methyl-³H]-thymidine incorporation and/or an increase in cell number in the NCI-N417 cells. We found that 10% FBS stimulated a 3.5-fold increase in [methyl-³H]-thymidine incorporation (fig. 14, left panel). The incorporation observed in the presence of [DPhe⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6-14) at either concentration was not significantly greater than that in unstimulated cells (fig. 14, left panel). The growth kinetic profile of NCI-N417 cells was examined using the MTS assay. The cells displayed a 24-hr lag phase followed by 48 hr of logarithmic growth, which was followed by steady-state growth (fig. 14, right panel). With 10% FBS at 1, 3 and 5 days after plating, the detected absorbance was significantly greater than the untreated control. [DPhe⁶,Ala¹¹, Phe¹³,Nle¹⁴]Bn(6-14), at concentrations of 30 nM and 1000 nM, did not significantly increase the detected absorbance compared with that in the untreated cells (fig. 14, right panel).

View larger version (23K): [in this window] [in a new window]   Fig. 14.   Effect of BRS-3 receptor activation on DNA synthesis and proliferation in NCI-N417 cells. Left panel) NCI-N417 cells were incubated with medium containing no peptide, 10% FBS or [DPhe6,Ala11,Phe13,Nle14]Bn(6-14) at the indicated concentrations with [3H]-thymidine for 24 hr. The minimal and maximal stimulated values were 4096 ± 418 cpm and 14888 ± 97 cpm, respectively. Data are means ± S.E. from three experiments performed in triplicate. NS: not significantly greater than untreated cells. Right panel) NCI-N417 cells were plated in RPMI-160 medium with 2% FBS. Cells were treated in the medium with or without [DPhe6,Ala11,Phe13,Nle14]Bn(6-14) at the indicated concentrations, or 10% FBS. On the indicated days, the tetrazolium dye MTS was added, and the absorbance at 490 nM was measured after 3 hr of incubation. Values are the means ± S.E. from three experiments using six determinations. * Significantly greater than untreated cells (P < .01). ** Significantly greater than untreated cells (P < .002).

	Discussion

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