©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Novel Gastrin Receptors Mediate Mitogenic Effects of Gastrin and Processing Intermediates of Gastrin on Swiss 3T3 Fibroblasts
ABSENCE OF DETECTABLE CHOLECYSTOKININ (CCK)-A AND CCK-B RECEPTORS (*)

Pomila Singh (§) , Azar Owlia , Rosario Espeijo , Bosong Dai (¶)

From the (1) Department of Anatomy and Neurosciences, The University of Texas Medical Branch, Galveston, Texas 77555

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have reported previously mitogenic effects of gastrin on several immortalized and neoplastic cell lines, including Swiss 3T3 fibroblasts. Receptor subtypes, cholecystokinin (CCK)-A and CCK-B, for a closely related peptide, cholecystokinin, were recently cloned. These studies were undertaken to investigate if CCK-A- and CCK-B receptors were perhaps mediating the mitogenic effects of gastrin on Swiss 3T3 cells. Receptor antagonists that inhibit the biological effects and binding of peptides to the CCK-A (L-364,718 (L18)) and CCK-B (L-365,260 (L60)) receptors were ineffective toward inhibiting the binding and proliferative effects of gastrin on Swiss 3T3 cells. Radiolabeled L18 and L60 demonstrated no binding to the cells, indicating that CCK-A and CCK-B receptors may be absent on Swiss 3T3 cells. Radiolabeled CCK-8, gastrin, L18, and L60, on the other hand, demonstrated specific binding to a pancreatic cancer cell line (AR42J cells) (used as a positive control). In cross-linking studies the molecular mass of the major band of gastrin receptors (GR) on Swiss 3T3 cells was determined to be 45 kDa. The mitogenic potency of 0.1-1.0 n M gastrin-like peptides on Swiss 3T3 cells was in the order of G1-17 G1-17-Gly > G5-17 G5-17-Gly > G2-17 > CCK-8-Gly G1-17-Lys CCK-8. The relative binding affinity of the peptides (based on the dose-dependent inhibition of binding of I-G1-17 to Swiss 3T3 cells) was similar to the relative mitogenic potency of the peptides as given above. Furthermore, G1-17-Gly was equally effective as G1-17 in displacing the binding of I-G1-17 to the 45-kDa GR from the Swiss 3T3 cells. Based on these studies it became evident that the novel gastrin preferring GR, expressed by Swiss 3T3 cells, binds and mediates the mitogenic effects of not only the mature (amidated) forms of gastrin-like peptides but also binds and meditates the mitogenic effects of glycine-extended forms of gastrin-like peptides.

Possible mRNA expression of CCK-A and CCK-B receptor subtypes by gastrin-responsive rodent intestinal and fibroblast cell lines (Swiss 3T3, IEC-6, CA) was measured by the methods of Northern blot analysis and reverse transcriptase-polymerase chain reaction. mRNA from rat pancreas, AR42J cells, and rat antrum served as positive controls. We were unable to detect CCK-A and CCK-B receptor mRNA in the three cell lines by both the methods. The positive control samples, on the other hand, gave the expected results. Thus the three gastrin-responsive rodent cell lines examined in this study lacked CCK-A and CCK-B receptors, but were positive for the gastrin preferring GR that demonstrated physicochemical and pharmacological attributes distinct from CCK-A and CCK-B receptors. Since colon cancers are known to express high concentrations of non-amidated gastrin-like peptides, a possible autocrine role of glycine extended forms of gastrin becomes a highly relevant clinical question in the light of the presence of novel GR on the fibroblast and intestinal cell lines.


INTRODUCTION

Several gut peptides hormones, including gastrin and bombesin (BBS),() are mitogenic for normal and cancerous intestinal mucosal cells (1, 2, 3, 4, 5) . In recent studies significant mitogenic effects of gastrin (6) and BBS (7) were also reported on a mouse fibroblast cell line, Swiss 3T3 cells. Although BBS receptors (BBS-R) have been identified and characterized on Swiss 3T3 cells (7) , gastrin receptors that may be mediating the mitogenic effects of gastrin on Swiss 3T3 cells remain to be identified. In the present study, binding characteristics of gastrin to Swiss 3T3 cells are presented for the first time.

Cholecystokinin (CCK) shares significant structural homology with gastrin at the C-terminal end and mediates its biological effects via CCK-A and CCK-B receptor subtypes (8) . CCK-A and CCK-B receptor subtypes were recently cloned from brain, stomach, and pancreatic cells (8, 9, 10, 11) . Recent reports suggest that CCK-A and CCK-B receptors can potentially mediate mitogenic effects of CCK/gastrin-like peptides on normal and cancerous pancreatic cells (12, 13, 14) and on small cell lung cancers (15) . It remains to be determined if CCK-A and CCK-B receptor subtypes are also expressed by normal and cancerous intestinal (IEC-6, CA) and fibroblast (Swiss 3T3) cell lines, which demonstrate a significant mitogenic response to gastrin and express high-affinity ( K= 0.1-1.0 n M) and low-affinity ( K= 0.1-1.0 µ M) forms of specific gastrin binding sites (6, 16) . In order to investigate this possibility we analyzed fibroblast (Swiss 3T3) and intestinal (CA and IEC-6) cell lines for the possible expression of CCK-A and CCK-B receptors by the methods of Northern blot and reverse transcriptase-polymerase chain reaction (PCR). At the pharmacological level, specific binding of radiolabeled gastrin, CCK-8, CCK-A receptor antagonist (L-364,718), and CCK-B receptor antagonist (L-365,260) were also measured. The growth stimulatory effects of gastrin were measured in the presence of CCK-A and CCK-B receptor antagonists in order to identify the receptor subtype that may be mediating the mitogenic effects of gastrin on the intestinal and fibroblast cell lines.

We and others have recently reported mitogenic effects of a processing intermediate of gastrin (glycine-extended gastrin) on human colon cancer (17) and pancreatic cancer (18) cell lines, suggesting for the first time that amidation of gastrin peptides is not a prerequisite for measuring mitogenic effects of gastrin-like peptides. In order to further confirm the mitogenic potency of noncarboxyamidated gastrins, we measured the binding and mitogenic potency of Gly- and Lys-extended forms of gastrins in comparison with that of amidated gastrins. Our results point to a possible presence of a novel class of gastrin preferring receptors that bind amidated and glycine extended forms of gastrin with almost equal affinity and are not recognized by the CCK-A and CCK-B receptor antagonists.


EXPERIMENTAL PROCEDURES

Cell Culture

The three cell lines used in this study included a mouse fibroblast cell line (Swiss 3T3), a normal rat intestinal epithelial cell line (IEC-6 cells) (American Type Culture Collection, Rockville, MD), and a mouse colon cancer cell lines (CA) (established in our laboratory (19) ). A subclone of Swiss 3T3 cells (that were enriched in GRP receptors and developed by Dr. A. Oliff) were obtained from Dr. E. Spindel (Division of Neuroscience Oregon Regional Primate Center). Swiss 3T3 cells were also obtained from ATCC at early passages of <35 and late passages of >100. All the cell lines were grown as monolayer cultures in either Dulbecco's modified Eagle's medium (IEC-6, Swiss 3T3) or RMPI 1640 (CA) (Life Technologies, Inc.), supplemented with glutamine (2 m M) and 10% heat-inactivated fetal calf serum (FCS, Hyclone, Logan, UT) in an atmosphere of 95% O, 5% COat 37 °C. The cell lines were regularly monitored for the absence of mycoplasma, using the fluorescence stain Hoechst 33258. Stock cultures of cells were subcultured at appropriate intervals to maintain the cells at subconfluent densities. For cell counting and subculturing the cells were dispersed with 0.05% trypsin and 0.02% EDTA.

In Vitro Growth Assays

Cell numbers were either measured directly by counting the total number of cells using a Coulter electronic particle counter (model ZF) or measured indirectly by using a semi-automated tetrazolium-based colorimetric assay (MTT assay). Both the MTT and cell counting assays were conducted essentially as described previously (20) . Briefly, for the MTT assay, cells (1-2 10cells/well) were plated into 96-well plates with 200 µl of normal growth medium containing 10% FCS. After 24 h, the medium was changed to serum-free medium, and the cells were cultured for 24 h in order to achieve quiescence. Cell were then stimulated with or without various doses of the peptides in the presence or absence of the nonpeptide receptor antagonist for 48-72 h in serum-free medium. At the end of the treatment, 0.1 mg (50 µl of 2 mg/ml) of MTT was added to each well and incubated at 37 °C for an additional 4 h. The medium was then aspirated from the well, leaving about 30 µl of medium in each well. One-hundred fifty µl of dimethyl sulfoxide (MeSO, Sigma) was added to each well, and the plates were shaken for 10 min to solubilize the formazan crystals. The plates were then read immediately at 540 n M on a scanning multiwell spectrophotometer (Umax, Molecular Devices, Melno, CA). Readings from wells containing culture medium alone with no cells, or cells without various drugs, were used as control. Optical absorbance readings from test wells were corrected against basal control absorbance values. In growth assays where the cell numbers were directly measured with the help of a Coulter counter, an optimal number of cells (0.5-1.0 10cells) were plated in 35-mm dishes with 2 ml of normal growth medium containing 10% FCS. After 24 h, the medium was changed to serum-free medium and cells cultured for an additional 24 h. Cells were then treated with various peptides ± receptor antagonists for 48 h in serum-free medium. At the end of the treatment, cells were disbursed with trypsin-EDTA solution as given above and cells counted using either a Coulter electronic counter or with the help of a hemacytometer under light microscope.

Binding of Radiolabeled Agonists and Receptor Antagonists with the Swiss 3T3 Cells

For measuring specific binding of gastrin to the Swiss 3T3 cells, we used either synthetic human gastrin (G1-17) or human gastrin 2-17 (G2-17) (Research Plus, Bayonne, NJ). G1-17 and G2-17 were iodinated with IODO-GEN (Pierce) as described previously (21) and specific activity calculated to be 1000-2000 cpm/fmol. For measuring specific binding sites for CCK, I-BH-CCK-8-sulfate (DuPont NEN) was used. For measuring specific binding sites for CCK-A and CCK-B receptor antagonists, H- L-365,260 (specificity activity of 160 dpm/fmol) and H- L-364,718 (specificity activity of 184 dpm/fmol) (DuPont NEN) were used, respectively. For cell binding assays, Swiss 3T3 cells were subcultured in 160-mm flasks and grown to subconfluence. All binding assays were performed 36-48 h after cell seeding in culture medium containing 10% FCS and 2% glutamine. Before the start of the binding assays, the cells in culture were washed with Hanks' balanced salt solution (HBSS, Life Technologies, Inc.) containing 0.1% bovine serum albumin (BSA) and 25 m M HEPES (Sigma) and scrapped with a rubber policeman into conical tissue culture polystyrene tubes. Cells were centrifuged at 500 g for 5 min and resuspended in HBSS at a concentration of 1-3 10cells/ml. Aliquots (0.5 ml) of suspended cells in polystyrene tubes were used in all binding assays. All binding assays were conducted essentially as described previously (gastrin (21) , CCK (22) , L-364,718, and L-365,260 (13, 23) ). Briefly, cells in suspension were incubated with either the I-labeled peptide (0.5-1.0 n M G1-17; 0.025 n M CCK-8) or the H-antagonist (2 n M) in the presence or absence of increasing concentrations (0.1 n M to 10.0 µ M) of either the homologous or the heterologous peptide/antagonist. The antagonists L-365,260 and L-364,718 were added at a final dilution of 0.01% MeSO in the binding assay tubes. All control and total binding tubes received the same concentration of MeSO. Nonspecific binding was determined in all assays in the presence of 1000-fold excess of the nonlabeled peptide/antagonist. Binding assays were performed generally at 37 °C for 30 min at pH 7.4 (determined to be optimal for most binding assays as described previously (21, 22, 23) ). At the end of the incubation, the cells were pelleted and washed twice with 1 ml of fresh ice-cold HBSS + 0.1% BSA. In the case of the peptide hormone assays, cell pellets were counted for I in a -counter (model 5500, Beckman) with 70% efficiency for I counting. In the case of assays for receptor antagonists, the cell pellets were counted for H using 10 ml of scintillation fluid, consisting of 5 g of permablend 2 (Packard Instruments) dissolved in 1 liter of toluene and counted in a scintillation counter (Packard TriCarb 460C, Downers Grove, IL) at <2% counting error with conversion to disintegrations/min by external standard ratio method. The binding affinity and total binding capacity of gastrin for binding the Swiss 3T3 cells were determined from a Scatchard plot of the specific binding data (21) . The relative binding affinity (RBA) of the amidated and non-amidated gastrin-like peptides for binding the high-affinity GR on Swiss 3T3 and CA cells was determined from a log-dose inhibition of specific binding of I-G1-17 by various peptides and receptor antagonists as described previously (21) .

Determination of the Molecular Mass of GR on Swiss 3T3

The molecular mass of GR on Swiss 3T3 cells was measured in cross-linking experiments as described previously (24, 25) . Aliquots of Swiss 3T3 cells (5 10/ml) were labeled with 1 n M I-gastrin 2-17 as given above in the presence of 0.1% soybean trypsin inhibitor, 0.1% bacitracin, and 100 units/ml of Trasylol and 1 m M phenylmethylsulfonyl fluoride and incubated with 1 m M disuccinimidyl suberate (DSS, Sigma) for 15 min at 22 °C, in the absence of BSA. DSS was dissolved in MeSO, immediately prior to use, and added to the substrate to give a final concentration of 1% MeSO in the binding buffer (HBSS, pH 7.4, containing 1 m M phenylmethylsulfonyl fluoride). The reaction was terminated by rapid centrifugation and the cells washed with excess binding buffer, devoid of BSA. The washed cell pellets were solubilized by boiling at 100 °C for 3-5 min in 0.2 M Tris HCl buffer, pH 6.8, containing 6% sodium dodecyl sulfate (SDS, w/v), 2 m M EDTA, 10% glycerol (v/v) in the presence or absence of 4% -mercaptoethanol (v/v). The supernatant was then subjected to SDS-polyacrylamide gel electrophoresis using 9.5% acrylamide in the separating gel and 4% acrylamide in the stacking gel. The gels were stained, destained, dried, and exposed to Kodak XAR-5 film for 5-7 days at -70 °C. The autoradiograms were densitometrically analyzed with the help of a laser densitometer (LKB UltroScan XL enhanced densitometer) and GelScan XL Software.

PCR and Northern Blot Analysis

Total cellular RNA was isolated as described previously (26) from various cell lines. RNA was recovered by ethanol precipitation and quantitated by its absorbance at 260 nm. Purity of RNA was determined by its absorbance at 260/270/280 nm, and the integrity was assessed by fractionating on 1% formaldehyde-agarose gels and observing the intensities of 28 and 18 S RNA bands after staining with ethidium bromide. Northern blot analysis of the samples was carried out as described previously (26) . Briefly the samples (30 µg of RNA) were electrophoresed through formaldehyde-agarose gels, transferred to a nylon membrane, and cross-linked with UV light. For analyzing the presence of CCK-A and CCK-B receptor mRNA, plasmids containing the full-length cDNA fragments for the rat CCK-A and CCK-B receptors were used which were kindly provided by Dr. Steven Wank. The rat cDNA probes used included: 1) a 1.5-kilobase pair XbaI insert of CCK-A receptor cDNA fragment inserted in pcDNA-1 plasmid and a 2.2-kilobase pair EcoRI insert of CCK-B receptor cDNA fragment subcloned in the vector PGEM (3ZF+), as per the information provided by Dr. Wank. The cDNA probes were labeled by nick translation and used for Northern hybridization of the RNA blots. The blots were washed under less stringent conditions (2 15 min at room temperature with 6 SSPE (1 SSPE, 0.18 M NaCl, 10 m M sodium phosphate (pH 7.7), 1 m M EDTA) 0.1% SDS, and 2 15 min at 48 °C with 1 SSPE, 0.1% SDS) in order to enable us to detect CCK-A and CCK-B receptor-like transcripts that may show only partial homology with the cDNA probes used.

cDNA Synthesis and PCR Amplification

cDNA synthesis and PCR was conducted as described previously (17) . For PCR amplification of the cDNA, 10 pmol each of upstream and downstream primers and 1 unit of AmpliTaq DNA polymerase (Perkin-Elmer) was added to the reaction mixture. The reaction mixture was subjected to 30 cycles of DNA polymerization (72 °C, 1.5 min), denaturation (94 °C, 30 s), and primer annealing (58 °C, 30 s) in a Perkin-Elmer DNA thermal cycler. PCR products were analyzed by electrophoresis on 1.5% agarose gels. The upstream and downstream primers were designed based on published cDNA sequences of rat CCK-A and CCK-B receptor genes in order to flank the coding sequences for the mature peptide. The sequences for the specific primers used are given in the legend of Fig. 7.


Figure 7: Detection of CCK-A and CCK-B receptor mRNA in various cell lines/tissues by Northern blot analysis ( A and B) and reverse transcriptase-PCR amplification ( C). Autoradiographs of representative Northern blots for CCK-A receptors ( A) and CCK-B receptors ( B) are shown from a total of four separate blots. Northern hybridization for CCK-A and CCK-B receptors was conducted under low stringency conditions using the appropriate P-labeled cDNA probes. A total of 30 µg of total RNA was loaded per lane. RNA samples in A in lanes 1-5 are: rat pancreas ( lane 1), AR42J cells ( lane 2), IEC-6 cells ( lane 3), CA tumors ( lane 4), and Swiss 3T3 cells ( lane 5). RNA samples in B in lanes 1-5 are: IEC-6 cells ( lane 1), CA tumors ( lane 2), Swiss 3T3 cells ( lane 3), rat pancreas ( lane 4), and AR42J cells ( lane 5). Arrows to the right of the figures ( A and B) point toward the hybridization of the probes to 2.7-kilobase transcript for CCK-A receptors ( A) and 2.7-kilobase transcript for CCK-B receptors ( B). Reverse transcriptase-PCR amplification of CCK-A and CCK-B receptor transcripts from various tissues/cell lines, stained with ethidium bromide on 1.5% agarose gels, is shown in C. cDNA was synthesized from 1 µg of total RNA and amplified by PCR for 25 (CCK-B receptor) to 30 (CCK-A receptor) cycles using the appropriate primer sets as described under ``Experimental Procedures.'' For amplification of CCK-A receptor cDNA, a 5`-3` 24-nucleotide sense oligomer from nucleotides 386 to 409 and an antisense 24 oligomer 5` to 3` from nucleotides 751 to 728 (based on the published sequence of rat CCK-A receptor cDNA (56)) were used to amplify a 366-bp cDNA fragment. For amplification of CCK-B receptor cDNA, a 5` to 3` sense 24 oligomer from nucleotides 794 to 817 and an antisense 24 oligomer 5` to 3` from nucleotides 1287 to 1264 (based on the published cDNA sequence of rat CCK-B receptors (11)) were used to amplify a 493-bp cDNA fragment. Samples in lanes 1-5 in C represent amplification with CCK-A receptor primers and samples in lanes 6-10 represent amplification with CCK-B receptor primers as shown. The samples in lanes 1-10 in C are: CA tumors ( lanes 1 and 6), Swiss 3T3 cells ( lanes 2 and 7), IEC-6 cells ( lanes 3 and 8), AR42J cells ( lanes 4 and 9), and rat pancreas ( lanes 5 and 10). The position of the molecular mass markers in base pair units is shown on the left-hand side of C. The arrows on the right-hand side of C point toward amplification of 1) a 409-bp CCK-A receptor cDNA fragment ( C), 2) amplification of a 493-bp CCK-B receptor cDNA fragment ( B), and 3) a 690-bp fragment ( A). The 690-bp fragment was amplified in the presence of the CCK-B receptor primers in all the samples, suggesting that the primers amplified contaminating gDNA in the samples which probably has a small 200-bp intron.



Statistical Analysis

Differences between means were compared by the use of the unpaired Student's t test and were considered statistically significant at the level of p < 0.05.


RESULTS

Proliferative Effects of Gastrin on Swiss 3T3 Cells in Vivo

The growth response of Swiss 3T3 cells to increasing concentrations of gastrin is presented in Fig. 1. The proliferation of Swiss 3T3 cells increased sharply in response to 0.5-1.0 n M gastrin and declined in the presence of higher concentrations (5-10 n M) of gastrin. A second smaller peak of activation was obtained between 0.1 and 0.5 µ M gastrin, which was once again reduced to control levels in the presence of 1-10 µ M gastrin. Gastrin at doses of 0.5-1.0 n M was most effective and increased the total number of Swiss 3T3 cells significantly above control values within 48 h of stimulation ( n = 8). The maximum growth stimulatory effect of 0.5 n M gastrin was measured to range from 40 to 110% over and above control values in different experiments. The proliferative effects of the maximum effective dose of insulin-like growth factor-I (IGF-I) and BBS (that are mitogenic for the subclone of Swiss 3T3 cells), were also measured simultaneously. Both IGF-I and BBS demonstrated a significant mitogenic effect at the maximum effective dose of 15 ng and 10 n M, respectively. IGF-I (2 n M) was only slightly more potent than 0.5 n M gastrin, and the difference was not statistically significant. BBS (10 n M), on an average, was slightly less potent than 0.5 n M gastrin, but once again the difference was not statistically significant. At higher doses, the mitogenic potency of IGF-I (6.6 n M) and BBS (50 n M) was similar to that measured at the maximally effective concentrations of the two peptides (data not shown). Similarly, in previous studies we have measured a plateauing in the growth response of colon cancer cells to BBS (4) and IGF-I (27) in response to increasing concentrations of the two peptides with no evidence of a sharp decline in the potency of the peptides at higher concentrations as measured in the case of gastrin.


Figure 1: Dose-dependent growth effects of gastrin, IGF-I, and bombesin on Swiss 3T3 cells. Swiss 3T3 cells in culture were stimulated with or without various concentrations of G1-17 and two optimally effective doses of IGF-I and bombesin as shown. Growth was measured in terms of optical density/well in an MTT assay as described under ``Experimental Procedures.'' The optical density values measured in control (nontreated) wells was arbitrarily assigned a zero value, and all changes in the OD measurements from the peptide-stimulated wells were converted as a percent value of the measurements in the control wells. Each bar graph represents mean ± S.E. values from six observations from a representative experiment; the asterisk = p < 0.05 versus control (0) value.



Binding Affinity and Molecular Mass of Gastrin Receptors on Swiss 3T3 Cells

In preliminary studies we established that specific binding of I-gastrin increased linearly with increasing doses of the radiolabeled ligand as we have reported previously for gastrin binding to colonic mucosal membranes (24) . Based on these preliminary results, it became evident that there were at least two classes of specific gastrin binding sites present on Swiss 3T3 cells, arbitrarily named Type I and Type II sites, just as we have reported previously for gastrin binding sites on normal and cancerous colonic cells (6, 16, 24, 28) . In order to determine if the binding affinity of the Type I and Type II sites on Swiss 3T3 cells was similar to that we have previously measured on other intestinal cell lines, the specific gastrin binding data was re-plotted as a Scatchard plot (Fig. 2 A), and equilibrium dissociation constants ( K) for the Type I and Type II sites calculated. Based on these calculations, the Kof Type I sites was determined to be 0.83 ± 0.56 n M ( n = 3) with a binding capacity of 48.3 ± 23.0 fmol/10cells. The total number of high affinity gastrin binding sites measured on Swiss 3T3 cells varied from 0.5 to 6.0 10sites/cell, depending upon the passage number of the cells, confluence of the cells in culture dish, and age of the cells in culture. The highest number of gastrin binding sites were measured in 70-80% confluent cells that were <30 days in culture and were taken from early passage numbers. A very large number of Type II sites, with a Kof 64.7 ± 22.0 n M, were measured (20-40). The total number of Type II sites measured was 1.19 ± 0.135 pmol/10cells.

The molecular mass of GR on Swiss 3T3 cells was determined by cross-linking of I-gastrin 2-17 to the Swiss 3T3 cells as described under ``Experimental Procedures,'' in the presence or absence of 1000-fold excess gastrin (Fig. 2 B). GR cross-linked to I-gastrin 2-17 moved as a single major band of 45 kDa. As can be seen from Fig. 2B, >90% of labeled gastrin bound to the 45-kDa receptor proteins could be displaced by excess nonlabeled gastrin. The autoradiographic profile was similar under reducing and nonreducing conditions, indicating that the 45-kDa binding proteins did not represent subunits linked by disulfide bonds. At longer exposure times of >14 days we could measure additional bands of 70-80 kDa and 32 kDa cross-linked to I-G2-17, which represented <10% of the total specific binding sites present on the Swiss 3T3 cells (data not shown).


Figure 2: A, Scatchard plot of specific binding of I-G1-17 to Swiss 3T3 cells. Swiss 3T3 cells were incubated with 0.5 n M I-gastrin-17 in the presence or absence of increasing concentrations of unlabeled gastrin as described under ``Experimental Procedures.'' The specific binding data, when analyzed by nonlinear regression analysis by the least squares method, demonstrated the presence of two classes of binding sites, labeled Type I and Type II sites. The specific binding data from the two sites was re-plotted as Scatchard plot with the x axis, indicating the femtomoles of specifically bound gastrin/10cells for the two sites. B, autoradiographic profile of I-G2-17 cross-linked to gastrin receptors on Swiss 3T3 cells. Swiss 3T3 cells were incubated with I-G2-17 ± 1000-fold excess unlabeled G1-17 and cross-linked with 1 m M DSS as described under ``Experimental Procedures.'' Solubilized membrane proteins were subjected to SDS-PAGE and analyzed autoradiographically. The data are representative of more than six experiments. Lanes 1 and 2 = total binding under nonreducing conditions; lanes 3-6 = binding under reducing conditions (+4% -mercaptoethanol); lanes 2, 5, and 6 = I-G2-17 + 1 µ M G1-17. The position of the molecular mass markers in kilodalton units is shown on the left-hand side of the figure. The arrow points to the 45-kDa gastrin-binding protein band that represented >90% of the specifically bound gastrin binding sites.



Effect of CCK-A and CCK-B Receptor Antagonists on the Proliferative Effects of Gastrin on Swiss 3T3 Cells

Since 0.5-1 n M gastrin demonstrated maximum growth stimulatory effects on Swiss 3T3 cells, we chose 0.5 n M gastrin as an optimal stimulatory dose for measuring possible inhibitory effects of CCK-A (L-364,718) (L-18) and CCK-B (L-365, 260) (L-60) receptor antagonists. Cells were stimulated with or without 0.5 n M gastrin in the presence or absence of increasing concentrations of L-18 and L-60 and the percent change in the cell numbers measured in a growth assay as described under ``Experimental Procedures.'' As can be seen from Fig. 3, neither L-18 or L-60 had any significant effect on the basal growth of the cells in the absence of gastrin. L-18 and L-60, at all doses examined, had no significant effect on the growth stimulatory effect of 0.5 n M gastrin. Surprisingly, 10 n M L-60, on an average, appeared to augment the growth of gastrin-stimulated cells, but the difference was not statistically significant.

Proliferative Effects of Amidated and Non-amidated Gastrin-like Peptides on CA, Swiss 3T3, and IEC-6 Cells

In order to measure the relative mitogenic potency of amidated and non-amidated gastrin-like peptides, we measured the proliferation of the fibroblast and intestinal cell lines in response to the physiological concentrations (0.1-10.0 n M) of the various peptides. Proliferative effects of gastrin-like peptides on Swiss 3T3 cells are presented in , and the proliferative effects of the peptides on CA and IEC-6 cells are presented in Table II. As can be seen from these results, in all cases glycine extended forms of gastrin were as effective as the amidated forms of gastrin in stimulating the growth of Swiss 3T3 () and intestinal cell lines (). Based on the results with Swiss 3T3 cells, the relative mitogenic potency of the various peptides examined was in the order of G1-17 = G1-17-Gly > G5-17 = G5-17-Gly > G2-17 > CCK-8-Gly CCK-8 = G1-17-Lys. Glycine-extended gastrins were similarly found to be equally effective as amidated gastrins in stimulating the growth of CA and IEC-6 cells (). In order to further compare the mitogenic potency of glycine extended gastrin and amidated gastrin, the growth response of Swiss 3T3 cells to the two peptides was compared at several doses ranging from 0.1 to 10.0 n M (Fig. 4). G1-17-Gly was only slightly less potent than G1-17 at doses <1.0 n M, but at doses 1.0-10.0 n M the growth response profile of Swiss 3T3 cells to the two peptides was almost identical. The lowest effective dose of the two peptides ranged from 0.1 to 0.25 n M, and the highest effective dose ranged from 0.5 to 1.0 n M, in three separate experiments. Thus the effective growth stimulatory concentrations of the gastrin-like peptides was within a narrow range of concentrations (1 log dose), with the stimulatory effects sharply declining thereafter. This was an intriguing phenomenon which has been measured with other biological effects of gastrin/CCK-like peptides and is certainly worthy of further investigation.


Figure 4: Dose-dependent proliferative response of Swiss 3T3 cells to G1-17 and G1-17-Gly. Swiss 3T3 cells in 35-mm Petri dishes were stimulated with or without increasing concentrations of the two peptides as shown, and the percent increase in the growth of the cells in treated dishes versus that in the control (arbitrarily assigned zero level) dishes was measured as described in the legend to Fig. 1. Each value is the mean ± S.E. of six observations from two separate experiments (1 and 2). In experiments 1 and 2, a total of 8.0 10cells and 6.0 10cells were seeded, respectively. At the end of the assay, on an average, 9.86 10and 7.8 10cells were measured in the control dishes of experiments 1 and 2, respectively (with <5% variation in individual values).



RBA of Gastrin-like Peptides and Receptor Antagonists for GR on Swiss 3T3 and CA Cells

In initial experiments we used tumor cell membranes prepared from CA tumors as substrate for the binding assays. Specific binding of I-gastrin in the presence or absence of increasing concentrations of various test peptides and receptor antagonists to the CA membrane aliquots was measured in a binding assay as described under ``Experimental Procedures.'' The binding of 1 n M I-gastrin was completely displaced (inhibited) in the presence of 100-1000-fold excess of nonlabeled gastrin. The percent inhibition of binding in the presence of various doses of the peptides and the receptor antagonists is shown in Table III. Glycine-extended gastrins were almost as effective as amidated gastrins in displacing the binding of 1 n M I-gastrin to the CA membranes. The CCK-A and CCK-B receptor antagonists (L-18 and L-60) were ineffective toward displacing the specific binding of I-gastrin to the CA membranes (I). Based on the dose-dependent displacement of the binding in the presence of increasing concentrations of the various peptides, the RBA was calculated as described under ``Experimental Procedures'' and was 100, 40, 8, 1.3, and 0.6 for the five peptides G1-17, G1-17-Gly, G5-17, CCK-8, and CCK-8-Gly, respectively (wherein RBA of G1-17 was arbitrarily assigned a value of 100). The receptor antagonists, L-18 and L-60, demonstrated no significant affinity for the specific gastrin binding sites on Swiss 3T3 cells (Fig. 5 A). In order to ascertain that the L-18 and L-60 being used by us were biologically effective, we used AR42J cells as a positive control. Both L-18 and L-60, as expected, displaced specifically bound radiolabeled CCK-8 to AR42J cells in a dose-dependent manner (Fig. 5 B). Since gastrin is known to primarily bind CCK-B receptor subtype on the AR42J cells, L-60 displaced 70% of the specifically bound radiolabeled gastrin to AR42J cells, whereas L-18 was not as effective (Fig. 5 C). The fact that L-60 did not displace 100% of the specifically bound G1-17 to the AR42J cells (even at 10times excess concentration) suggested that 30% of specific gastrin binding sites on AR42J cells were perhaps distinct from CCK-B receptors (and may be similar to the gastrin preferring receptors on the intestinal and Swiss 3T3 cells (6, 16, 29, 30) ). G1-17-Gly was once again as effective as amidated gastrin in displacing the binding of radiolabeled gastrin to Swiss 3T3 cells (Fig. 5 A). Unlike the CA cells, however, the RBA of G1-17-Gly was almost identical to the RBA of G1-17 for GR on Swiss 3T3 cells (Fig. 5 A). Since the majority of the gastrin binding sites on Swiss 3T3 cells appeared to be present on the 45-kDa protein, we measured the relative potency of amidated and non-amidated gastrin-like peptides for displacing the binding of 1 n M I-G2-17 cross-linked to the 45-kDa protein. In Fig. 6 A is shown an autoradiogram demonstrating the presence of 45-kDa GR proteins cross-linked to I-G2-17 in the presence or absence of increasing concentrations of the various peptides. Autoradiographic data from four separate blots were densitometrically analyzed and is presented in Fig. 6 B. As can be seen from this figure, G1-17-Gly was as effective as G1-17 in displacing the binding of radiolabeled G2-17 from the 45-kDa GR protein on Swiss 3T3 cells. At concentrations of 10 and 100 n M, G5-17-Gly was significantly less effective compared with G1-17. At all concentrations, CCK-8 was significantly less effective than G1-17. Interestingly CCK-8 at 10 and 100 n M concentrations appeared to augment the binding of G2-17, but the difference was not statistically significant.


Figure 5: RBA of the CCK receptor antagonists for gastrin and CCK binding sites on Swiss 3T3 and AR42J cells. Specific binding of I-G1-17 ( A, B) or I-BH-CCK-8 ( C) to Swiss 3T3 ( A) and AR42J ( B, C) cells was measured in the presence or absence of increasing concentrations of either the homologous peptide or the receptor antagonists L-18 and L-60 as described under ``Experimental Procedures.'' The total specific binding of radiolabeled ligands was arbitrarily assigned 100% value, and the displacement of the specifically bound counts in the presence of the indicated concentrations of the test substances was calculated as a percent inhibition of specific binding as described in Table IV. From the linear part of the log-dose inhibition curve, the RBA of the peptides and nonpeptide receptor antagonists was calculated as described previously (21). On the x axis the log molar concentration of the peptides/nonpeptide receptor antagonist is shown for Fig. 4, A-C. Additionally the molar excess concentration of the unlabeled peptide/antagonist used for displacing the binding of either 1 n M I-G1-17 (Fig. 4, A and B) or 10 p M I-BH-CCK-8 (Fig. 4 C) is also indicated. Each data point in Fig. 4 A represents mean ± S.E. of six independent measurements from two separate experiments. The data are representative of several similar experiments. Data in Fig. 4, B and C, are from a representative experiment of a total of two to three similar experiments, and each data point is the mean of four measurements, wherein the variation was <10% between the separate measurements.



Expression of CCK-A and CCK-B Receptors at the Protein Level by Swiss 3T3 and AR42J Cells

In order to measure the presence of specific binding sites for the CCK-A and CCK-B receptor subtypes, we chose the direct approach of measuring specific binding of the agonists (I-G1-17 and I-BH-CCK-8) and the receptor antagonists (H-L-18 and H-L-60) by the two cell lines. AR42J cells were chosen as a positive control, since these cells are known to express significant concentrations of both CCK-A and CCK-B receptors (8) . As can be seen from Table IV, both Swiss 3T3 cells and AR42J cells demonstrated the presence of specific binding sites for the agonists. However, specific binding sites that could recognize the radiolabeled antagonists were not detected on Swiss 3T3 cells but were detected in significant concentrations on the AR42J cells. These results thus further confirmed the possibility that the gastrin/CCK-like peptides bind to a unique or novel class of GR on Swiss 3T3 cells that are apparently distinct from the CCK-A and CCK-B receptors.

Expression of CCK-A and CCK-B Receptor mRNA by Fibroblast and Intestinal Cells

To further confirm the absence of expression of CCK-A and CCK-B receptors by Swiss 3T3 cells and other gastrin-responsive intestinal cell lines, RNA from several cell lines and tissues was screened for the possible presence of CCK-A and CCK-B receptor mRNA by the methods of Northern blot analysis (Fig. 7, A and B) and reverse transcriptase-PCR (Fig. 7 C), as described under ``Experimental Procedures.'' In all cases RNA from rat antrum, rat pancreas, and AR42J cells were analyzed as positive controls. As can be seen from Fig. 7 , A and B, the cDNA probes for the CCK-A and CCK-B receptors hybridized with the expected transcript sizes for the two receptors in the positive control samples (pancreas, AR42J cells). Hybridization with CCK-A and CCK-B receptor cDNA was not detected in either the intestinal cell lines (IEC-6, CA) or the Swiss 3T3 fibroblasts (Fig. 7, A and B). We next used the highly sensitive method of reverse transcriptase-PCR to detect possible presence of a low number of copies of the CCK-A and CCK-B receptors in the three test cell lines (CA, Swiss 3T3, IEC-6). Once again we were unable to measure an amplification of the CCK-A and CCK-B receptor message in either of the three cell lines (Fig. 7 C). The positive control samples, on the other hand, gave the expected results for the CCK-A and CCK-B receptors. In the absence of reverse transcriptase no amplification of the receptor message was measured in AR42J cells (data not shown), confirming amplification of cDNA in these samples. In the presence of the primers for CCK-B receptor cDNA, we additionally measured a weak band of 690 bp in all the samples which probably represented amplification of gDNA (which is usually present as <1% contaminant in RNA samples, untreated with DNase (17) ). This suggests that the gDNA for rat CCK-B receptor has at least one small intron of 200 bp. The sense and antisense primers for CCK-B receptors used by us for amplification of cDNA were also used for PCR amplification of gDNA from the same cell lines. Once again we measured amplification of a 690-bp gDNA fragment from all the cell lines (data not shown), indicating that a similar CCK-B receptor gene was present in all the cell lines examined. cDNA expression, however, could only be detected in the positive control samples, confirming the absence of expression of CCK-B receptors mRNA by the intestinal and fibroblast cell lines.

Based on the above results, it thus appears more than likely that the Swiss 3T3 fibroblasts and the intestinal cell lines do not express detectable levels of the CCK-A and CCK-B receptor subtypes and perhaps express novel GR that bind and mediate the mitogenic effects of gastrin/CCK-like peptides on these cells.


DISCUSSION

We have described for the first time the presence of gastrin-preferring receptors (GR) on an immortalized fibroblast cell line (Swiss 3T3 cells) that do not discriminate between amidated and glycine-extended gastrins and apparently mediate mitogenic effects of gastrin-like peptides on these cells. The early passages of Swiss 3T3 cells (<30), unlike the latter passage (>110), were highly responsive to BBS, insulin-like growth factors (IGFs), and gastrin (present studies). A subclone of Swiss 3T3 cells, enriched in cells expressing gastrin-releasing peptide (GRP) receptors (Dr. A. Oliff) were similarly highly responsive to BBS and gastrin (present studies). Swiss 3T3 cells demonstrated a dual (two peak) bi-phasic response to gastrin. Cells were maximally responsive to 0.5-1.0 n M gastrin and slightly responsive to 0.1-1.0 µ M gastrin. Some mouse and human colon cancer cells similarly demonstrate a dual bi-phasic response to gastrin (6, 16, 28, 29) . Several other human colon cancers, however, respond only to high concentrations of gastrin (17, 31) . High-affinity ( K= 1 n M) and/or low-affinity ( K= 0.1 µ M) gastrin binding sites are present on normal and cancerous intestinal cell lines (6, 16, 28, 31, 32) and Swiss 3T3 cells (present studies), arbitrarily termed Type I and Type II sites. Since gastrin was mitogenic within the dose ranges compatible with binding Type I and Type II sites, occupation of Type I and Type II sites may result in the two peak mitogenic effects of gastrin. Endogenous and recombinant CCK receptors exist in multiple binding states (23, 33) . Glycosylation (34) and association with G proteins and guanyl nucleotides (35, 36) are believed to effect the affinity status and/or ligand specificity of CCK receptors. It remains to be seen if Type I and Type II GR on Swiss 3T3 cells are different binding states of the same receptor or are two separate receptor subtypes.

The majority of the biological effects of gastrin-like peptides on normal target cells in the pancreas, gallbladder, stomach, and central nervous system are believed to be mediated by CCK-A and CCK-B receptors (8) . Transformed and cancerous cells arising from the pancreas and ECL cells also express high concentrations of CCK-B and/or CCK-A R receptors (12, 13, 37, 38) . Since the cloning of CCK-A and CCK-B receptors, however, none of the reports to date, to the best of our knowledge, have described the expression of CCK-A- and/or CCK-B-R by the normal and cancerous intestinal cells. There is, however, a large volume of literature from several laboratories including ours, wherein trophic effects of gastrin have been described on normal (1, 39, 40, 41) and cancerous (2, 5, 6, 20, 28, 29, 31) intestinal cells from both humans and rodent species. Based on structural requirements of gastrin-like peptides for binding GR on colon cancer cells, we reported a decade ago about the possible presence of unique GR (42) . Since then we have published several studies, all suggesting the presence of unique GR on normal and cancerous intestinal cells (6, 16, 17, 24, 25, 41) . The present studies confirm the presence of novel gastrin-preferring receptors on Swiss 3T3 and intestinal cells as discussed below.

The selective CCK-A (L-364,418) (L-18) and CCK-B (L-365,260) (L-60) receptor antagonists had no effect on either the mitogenic potency or the binding of gastrin to Swiss 3T3 and intestinal cells (CA and IEC-6). Even though multiple binding states of CCK receptors have been described (23, 33) , all the multiple affinity states of CCK-A-R bind L-18 with equal affinity (23) . Thus our results with the Swiss 3T3 and intestinal cells suggest that gastrin-preferring receptors do not meet the pharmacological criteria of CCK-A- and CCK-B-R. No specific binding sites for the two selective antagonists were measured on Swiss 3T3 cells further confirming this possibility. Additionally we did not detect mRNA for CCK-A- and CCK-B-R in the fibroblast and intestinal cell lines by either the reverse transcriptase-PCR method or by Northern blot analysis after low stringency washings; the positive control samples gave the expected results. Although we did not detect CCK-B-R mRNA in rat pancreas by the reverse transcriptase-PCR method (Fig. 7 C), we did detect a 2.7-kilobase band hybridized to the CCK-B-R cDNA in the Northern blots of rat pancreatic samples. We believe this band represents CCK-A-R mRNA and not CCK-B-R mRNA, since the two have 54% homology (8) and can be expected to hybridize under the low stringency conditions of washing. Since gastrin is mitogenic for human colon cancers (2, 5, 6, 16, 29, 31, 32) , Northern blots containing 5 µg of poly(A)RNA from representative human colon cancer cell lines (Colo-320, CaCo2) (that are responsive to gastrin in vitro) were analyzed by Dr. Kopin at our request. The gastrin-responsive human colon cancer cells were analyzed to be devoid of CCK-B-R mRNA. We have additionally utilized sense and antisense primer sets from the most conserved portions of the CCK-A- and CCK-B-R transmembrane domains and have once again failed to detect significant amplification of PCR products from either the normal or the cancerous intestinal cells from rat and human (data not shown). These results suggest that the novel class of gastrin-preferring receptors, expressed by the fibroblasts and normal and cancerous intestinal cell lines, may not belong to the family of G-protein-coupled receptor superfamily. In recent years several other investigators have similarly concluded that trophic effects of gastrin on colon cancer cell lines are probably mediated by a separate class of gastrin-preferring receptors that do not meet the criteria of either CCK-A- or CCK-B-R (29, 31, 32, 35, 43, 44, 45) . Nonspecific inhibitory effects of receptor antagonists at high concentrations, however, have been reported that are independent of binding CCK receptors (32, 43, 44) .

Secretion of prepro, non-amidated forms of gastrin by human colon cancers and colon cancer cell lines was recently reported (discussed in Ref. 17). High serum concentrations of non-amidated, prepro forms of gastrin are measured in serum under diseased states (reviewed in Ref. 46). We recently reported mitogenic effects of glycine-extended gastrins (G1-17-Gly) on colon cancers (17, 47) . In the present study we measured the relative mitogenic potency of several amidated and non-amidated gastrin-like peptides and the relative binding affinity of the peptides for GR on fibroblast and intestinal cells. The relative binding affinity of the amidated and non-amidated gastrin-like peptides closely resembled the relative mitogenic potency of the peptides. It thus became evident that glycine-extended forms of gastrin/CCK-like peptides were equally effective as their amidated counterparts, suggesting that the novel gastrin receptors did not discriminate between the glycine-extended and the amidated gastrin-like peptides. It was recently reported that G1-17-Gly binds a unique class of receptors that do not bind amidated gastrins and is apparently specific for binding the C-terminal Gly moiety (18) . Our results, however, suggest that yet another unique class of GR is expressed by some of the target cells that bind amidated and non-amidated gastrin-like peptides with almost equal affinity. G1-17-Lys had a significantly lower potency compared with G1-17-Gly, for displacing bound gastrin and for exerting mitogenic effects on Swiss 3T3 cells. Since gastrin is an acidic peptide, any modifications resulting in availability of a basic amino group for binding Glu residues could potentially result in altering the structure of the peptide which may explain the results with G1-17-Lys.

In earlier studies a possible autocrine role of gastrin-like peptides was speculated (48) . We and others (discussed in Refs. 17, 20, and 32) have since reported that amidated forms of gastrin are not likely to play an autocrine role in colon cancers. However, the observations of a mitogenic potency of G1-17-Gly (present studies and Refs. 17 and 18) raises the question of a possible autocrine role of processing intermediates of gastrin, which needs to be examined.

Utilizing I-G2-17 as the ligand GR of several molecular sizes, ranging from 30 to 200 kDa have been described (24, 49) . A predominant 75-80-kDa protein was measured on the parietal/fundic cells (50, 51) . On rodent and human colon cancer cells a 32-kDa GR protein was predominately measured (25) . In the present studies, GR on Swiss 3T3 cells were predominately 45 kDa in size, similar to that on colonic mucosal membranes from rodent species (24) . The 45-kDa protein on colonic mucosal membranes was reduced to an molecular mass of 32 kDa on deglycosylation (24) . The 45-kDa GR on Swiss 3T3 cells was also glycosylated and on digestion with endo-- N-acetylglucosaminidase F reduced to 30 kDa.() The molecular masses of the deglycosylated protein core of CCK-A and CCK-B receptors from different species and tissues have been reported to range from 37 to 42 kDa (discussed in Ref. 52), which is close to the theoretical mass (50 kDa) of the cloned proteins (8) . The cloned CCK-A- and CCK-B-R universally contain three to four potential sites for N-glycosylation (8) . CCK-A-R are heavily glycosylated (53) , whereas CCK-B-R are variably glycosylated (discussed in Ref. 52). CCK-B-R from calf pancreas (52) have an molecular mass similar to that of GR on Swiss 3T3 cells (present studies). The major difference, however, is in the pharmacology of the two receptors. CCK-B-R from the calf pancreas demonstrate an equal binding affinity for the agonists CCK-9 and G1-17 and a relatively high binding affinity for the CCK-B receptor antagonist L-60, whereas GR on Swiss 3T3 cells and CA cells demonstrate 10 times higher affinity for G1-17 versus CCK-8 and do not bind L-60 (present studies).

In order to further understand the role of gastrin-preferring receptors in proliferation of normal and cancerous intestinal mucosal cells, we will clearly need to further characterize the 45-kDa GR measured on these cells (Refs. 24 and 25 and present studies). Swiss 3T3 cells should provide a useful source for further purification and cloning of the cDNA for the unique and novel gastrin-preferring receptors.

  
Table: Proliferative effects of gastrin-like peptides on Swiss 3T3 cells

In two experiments (arbitrarily termed 1 and 2), 5 10and 7 10cells were seeded in each 35-mm dish. At the end of the growth assay, on an average 5.51 10and 7.99 10cells were measured in control dishes of experiments 1 and 2, respectively, with <5% variation in individual values within an experiment. Values are a mean ± S.E. of eight observations from the two experiments. Cells measured in control wells were arbitrarily assigned a 100% value. Cell numbers in peptide treated wells are expressed as a percent of cells in the control (nontreated) wells. Letters a-h indicate that the values are significantly different ( p < 0.05) compared with the indicated values for the corresponding dose. In order to ensure that increase in cell number reflected proliferation of cells, nuclear labeling of the cells with [H]thymidine was measured in a few control and treated dishes (treated with G1-17). Only 5-10% of cells demonstrated nuclear labeling in control dishes, whereas 30% of the cells treated with 0.5 and 1.0 n M gastrin were labeled, when examined autoradiographically (data not shown).


  
Table: 191

  
Table: Percent inhibition of I-gastrin (1 n M) binding to CA cells by gastrin-like peptides and nonpeptides antagonists


  
Table: +++


FOOTNOTES

*
This work was supported by Grant CA 38651 from the National Institutes of Health. Some of the information presented in this paper has been published in its preliminary form as abstracts (Refs. 6, 12, and 44). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom all correspondence and reprint requests should be addressed: Dept. of Anatomy and Neurosciences, 10.138 Medical Research Bldg., 1043, University of Texas Medical Branch, Galveston, TX 77555-1043. Tel.: 409-772-4842; Fax: 409-772-1861.

Visiting scientist from Tianjin China.

The abbreviations used are: BBS, bombesin; BBS-R, bombesin receptor; CCK, cholecystokinin; CCK-A-R and CCK-B-R, cholecystokinin A and B receptor(s), respectively; PCR, polymerase chain reaction; FCS, fetal calf serum; MTT assay, semi-automated tetrazolium-based colorimetric assay; HBSS, Hanks' balanced salt solution; BSA, bovine serum albumin; RBA, relative binding affinity; GR, gastrin receptors; DSS, disuccinimidyl suberate; IGF, insulin growth factor; bp, base pair(s); L18, L-364,718; L60, L-365,260; gDNA, genomic DNA.

P. Singh, A. Owlia, R. Espeijo, and B. Dai, unpublished data.


ACKNOWLEDGEMENTS

Gastrin 1-17-glycine, gastrin 1-17-lysine, and CCK-8-glycine were kindly provided to us by Dr. Jens Rehfled (Professor, Rigshospitalet, Department of Clinical Chemistry, Blegdamsvej 9, Copenhagen, Denmark). Gastrin 5-17-glycine was obtained from Dr. John DelValle (Assistant Professor, Department of Medicine, University of Michigan, Ann Arbor, MI). L-364,718 (3-(acylamino)-benzodiazepine analogue) and L-365,260 (3-(benzoylamino)-benzodiazepine analogue) were obtained from Dr. Roger M. Freidinger (Senior Scientist, Department of Medicinal Chemistry, Merck Sharp and Dohme, West Point, PA). We gratefully acknowledge the secretarial help of Kay Smith.


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