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The Journal of Clinical Endocrinology & Metabolism Vol. 85, No. 11 4131-4137
Copyright © 2000 by The Endocrine Society

Original Studies

Expression of the Calcium-Sensing Receptor in Gastrinomas

Stephan U. Goebel, Paolo L. Peghini, Paul K. Goldsmith, Allen M. Spiegel, Fathia Gibril, Mark Raffeld, Robert T. Jensen and Jose Serrano

Digestive Diseases Branch (S.U.G., P.L.P., F.G., R.T.J., J.S.) and Metabolic Diseases Branch (P.K.G., A.M.S.), National Institute of Diabetes and Digestive and Kidney Diseases, and Hematopathology Section, Laboratory of Pathology, National Cancer Institute (M.R.), National Institutes of Health, Bethesda, Maryland 20892

Address all correspondence and requests for reprints to: Dr. Robert T. Jensen, Digestive Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, Building 10, Room 9C-103, 10 Center Drive, MSC 1804, Bethesda, Maryland 20892-1804.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Extracellular calcium levels are able to influence the secretion of gastrin by gastrinomas and possibly affect the growth pattern. The molecular mechanisms of these functions are not known. The purpose of the present study was to investigate the presence of the calcium-sensing receptor (CaR) in 10 gastrinomas and determine the extent of expression in the tumors. The amounts of CaR messenger ribonucleic acid in eight tumors were determined by quantitative RT-PCR. Protein expression was analyzed by Western blot and immunohistochemistry using a monoclonal antibody (ADD). CaR messenger ribonucleic acid was detected in all gastrinomas with levels ranging from 0.04–3.16 times the amount of ß-actin transcripts. The Western blot showed a major immunoreactive band at 250 kDa and a minor at 140 kDa, corresponding to the receptor dimer and monomer, respectively. Immunohistochemistry demonstrated variable membranous staining in all gastrinomas and normal pancreatic islets. No staining was observed in the normal liver, lymph node, or exocrine pancreas. We conclude that the CaR is present in all gastrinomas, with expression varying by 80-fold. It probably contributes to the calcium-stimulated gastrin release by gastrinomas. Whether the density of the CaR is a determining factor of the magnitude of this gastrin release or plays a role in regulating the growth pattern of the gastrinoma, as it does in other cells, remains unclear at present.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
NUMEROUS STUDIES have demonstrated that calcium levels can have a profound effect on the biological behavior of gastrinomas (1, 2, 3, 4, 5, 6, 7) as well as other pancreatic endocrine tumors (insulinomas and vasoactive intestinal polypeptide-secreting tumors) and carcinoid tumors (5), which closely resemble pancreatic endocrine tumors (8). Increased extracellular calcium levels have a marked stimulatory effect on gastrin release by gastrinomas both in vivo and in dispersed isolated cells (4, 5, 6, 9, 10, 11). Similarly, calcium levels affect peptide release by other pancreatic endocrine tumors (9, 10). The effect of calcium levels on hormone release by these tumors has important clinical implications. This observation has been used clinically to diagnose gastrinomas with the calcium provocative test (3, 11, 12) and for functional tumor localization of various pancreatic endocrine tumors by assessing hormone gradients after selective intraarterial calcium injection (13, 14, 15).

A number of studies (1, 16) demonstrate that both basal gastrin levels and secretin-stimulated gastrin release from gastrinomas are affected by the presence or absence of hypercalcemia. In patients with Zollinger-Ellison syndrome and multiple endocrine neoplasia type 1 (MEN1) gastrin levels are altered by the level of activity of the frequently present primary hyperparathyroidism (1, 16, 17, 18). Furthermore, some evidence suggests that the hyperparathyroidism in patients with Zollinger-Ellison syndrome and MEN1 may contribute to the growth of the pancreatic endocrine tumor (19).

The exact mechanism of action of extracellular calcium in gastrinoma cells, other pancreatic endocrine tumors, carcinoid tumors, or other endocrine cells remains unclear. Voltage-gated calcium channels have been found to mediate calcium influx into these cells (20). However, in dispersed insulinoma cells, nifedipine, an inhibitor of voltage-dependent calcium channels, did not block all the effects of hypercalcemia (21). Recently, a calcium-sensing receptor (CaR) has been cloned, which is a 1085-amino acid protein that belongs to the G protein-coupled superfamily of 7 transmembrane domain receptors (22). This receptor regulates PTH release from parathyroid cells in response to varying extracellular calcium concentrations (23). The current study was designed to examine whether this receptor was present in gastrinomas and thus could possibly mediate some of the important clinical effects of calcium on these cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patients

Ten patients who underwent exploratory laparotomy for Zollinger-Ellison syndrome at the NIH between 1990 and 1998 were included in this study. The 10 patients included 8 patients who had tissue frozen and thus were suitable for competitive PCR analysis and two patients in whom normal liver and liver metastases were both present on paraffin sections. The study protocol was approved by the clinical research committee of the NIDDK, and all patients gave informed consent. The diagnosis of Zollinger-Ellison syndrome was established as previously reported (24). Serum gastrin levels were analyzed by RIA by Bioscience Laboratories (New York, NY) or Mayo Clinic Laboratories (Rochester, MN). The presence of MEN1 was diagnosed by a family history or laboratory evidence of other endocrinopathies on yearly evaluation, as described previously (25). Duration of disease was defined by the clinical history from the time of symptom onset as previously described (26). All patients underwent an exploratory laparotomy with an extensive intraoperative evaluation for attempted curative resection (27, 28). The patients were then reassessed within 2 weeks of surgery and 3–6 months postoperatively to determine cure and annually to monitor for progression of disease as previously described (24, 29). Based on serial imaging studies, the growth of tumors was classified as no growth if no new lesions developed and no increase in size occurred over the follow-up period. If there was an increase in tumor size or number, the tumor was classified as growing. If a more than 50% increase in volume over 6 months occurred, the tumor was classified as showing rapid growth, as defined previously (29).

Tumors

Competitive PCR. Tumor samples were immediately snap-frozen in liquid nitrogen during surgery and stored at -70 C. Tumor ribonucleic acid (RNA) was extracted from 8-µm cryosections of the specimens using a commercial kit (RNeasy Mini Kit, QIAGEN, Santa Clarita, CA) after analyzing an adjacent slide with hematoxylin and eosin staining to determine that there was no contamination with normal tissue. Random hexamer-primed first strand complementary DNA (cDNA) was prepared with RT (RNA PCR kit, Perkin-Elmer Corp., Foster City, CA). After RT, PCR was carried out for amplification of a 461-bp fragment of the human calcium-sensing receptor with the following primers: sense (CaR-S), 5'-AAGCACCTACGGCATCTAA-3' (nucleotides 1384–1402); and antisense (CaR-AS), 5'-GCGATCCCAAAGGGCTCCG-3' (nucleotides 1826–1844; modified from Ref. 30). PCR was carried out in a final volume of 25 µL with 0.5 µU DNA-polymerase (AmpliTaq Gold, Perkin-Elmer Corp.) and dimethylsulfoxide in a final concentration of 5%. The PCR reaction for the calcium-sensing receptor was run under the following conditions: initial denaturation at 94 C for 10 min, 40 cycles of 94 C for 50 s, 58 C for 50 s, 72 C for 50 s, and final extension at 72 C for 5 min in a thermal cycler (Perkin-Elmer Corp., 9700 thermocycler). A 626-bp fragment of ß-actin was amplified with the following primers: sense (ß-actin-S), 5'-CCTCGCCTTTGCCGATCC-3'; and antisense (ß-actin-AS), 5'-GGATCTTCATGAGGTAGTCAGTC-3', using the PCR-conditions as above, except with an annealing temperature of 60 C (31). The PCR product from both the mimic and native cDNA were sequenced and shown to contain the correct sequences.

For the quantification of CaR messenger RNA amounts a 675-bp fragment of genomic DNA was amplified to generate a CaR mimic. The following primers were used: sense (CaR-MIM-S), 5'-AAGCACCTACGGCATCTAAATCGACGACGTGGTGCGCCTGTTTG-3'; and antisense (CaR-MIM-AS), 5'-GCGATCCCAAAGGGCTCCGGAGGTGAGGTTGATGATTTGGAG-3', using the CaR PCR conditions. For the quantification of ß-actin messenger RNA (mRNA), a 488-bp fragment of genomic DNA was amplified to generate a ß-actin mimic. The following primers were used: sense( ß-actin-MIM-S), 5'-CCTCGCCTTTGCCGATCCCTTGCTCTCACCTTGCTCT-3'; and antisense (ß-actin-MIMAS), 5'-GGATCTTCATGAGGTAGTCAGTCTCTTCATCTGCACTTGCGAC-3', using the ß-actin PCR conditions. Stock solutions were prepared by purifying the PCR solutions with a Microcon 30 filter (Amicon, Inc., Beverly, MA). The specificity of all fragment amplifications was verified by automated sequencing (ABI Prism 377 DNA Sequencer, Perkin-Elmer Corp.). The concentrations of the mimic stock solutions were determined by measuring the optical density at 260 nm in a spectrophotometer (Beckman Coulter, Inc., Columbia, MD), and serial dilutions were prepared. Competitive PCR was performed by adding serial mimic dilutions to the target cDNA with the respective primer pairs (CaR-S/AS or ß-actin-S/AS) under the appropriate PCR conditions. The concentration of target cDNA was calculated by comparison to the concentration of the mimic, as seen by equal intensity of ethidium bromide staining in a 1% agarose gel. The results of the competitive PCR were expressed as the ratio of the number of molecules of the CaR mRNA to ß-actin mRNA present.

Western blotting. Fifty milligrams of tumor tissue were lysed in 0.5 mL lysis buffer [50 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 0.1% NaN3, 1 mmol/L ethyleneglycol-bis-(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 0.4 mmol/L ethylenediamine tetraacetate, 1% Triton X-100, 1% deoxycholate, and protease inhibitors (Complete Mini, Roche Molecular Biochemicals, Indianapolis, IN)]. One sample had 100 mmol/L iodoacetamide added to the lysis buffer. The protein concentration was measured by Coomassie Blue G250 dye (Bradford assay). Four micrograms of protein were mixed with SDS-gel loading buffer (loading solution 2x, Quality Biological, Inc., Gaithersburg, MD) in the presence or absence of 50 mmol/L ß-mercaptoethanol and loaded into a 4–12% Tris/glycine SDS-polyacrylamide gel. Proteins were separated by electrophoresis and transferred to nitrocellulose membranes (Protran, Schleicher & Schuell, Inc., Keene, NH). The membranes were blocked overnight at 4 C with 5% nonfat dry milk [Tris-buffered saline and 0.1% Tween-20 (TBS-T)] and incubated with the monoclonal antibody (1:10,000; ADD; directed at amino acids 214–235 of the CaR) (32) for 90 min in TBS-T. Membranes were washed in TBS-T for 10 min three times and incubated with peroxidase-linked sheep antimouse at 1:2,000 (Amersham Pharmacia Biotech, Piscataway, NJ) in TBS-T for 60 min. After washing the membranes in TBS-T for 10 min three times, the bands were detected using the enhanced chemiluminescence kit (Supersignal, Pierce Chemical Co., Rockford, IL) according to the manufacturer’s instructions.

Immunohistochemistry. Immunohistochemical staining was performed on an automated immunostainer (Ventana Medical Systems, Inc., Tucson, AZ) according to the company’s protocols, with slight modifications. Briefly, 8-µm thick paraffin sections were mounted on charged glass slides. After deparaffinization and rehydration, the slides were placed in a microwave pressure cooker containing 1500 mL 0.01 mol/L citrate buffer (pH 6.0) containing 0.1% Tween-20 and heated in a microwave oven at maximum power (900 watts) for 40 min. Sections were immediately cooled in Tris-buffered saline (0.05 mol/L; pH 7.6) containing 5% goat serum (Life Technologies, Inc., Grand Island, NY) for 30 min. In preliminary studies slides were incubated with the primary antibody (ADD) at dilutions of 1:100, 1:500, and 1:1000 for 12, 24, 36, and 48 h at room temperature. The conditions of a 1:500 dilution for antibody and 36-h incubation gave the best results and were used in the study. The rest of the procedure (secondary antibody, avidin-biotin complex, color development, and counterstain) was performed on the Ventana immunostainer using the standard procedures recommended.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
A total of 10 gastrinomas from 10 patients were studied. Clinical characteristics of the 10 patients are summarized in Table 1Go. This cohort is similar to other surgical series (4, 33, 34, 35) with respect to patient’s gender, mean age of patients (46 yr), and duration of disease, as defined by the time from the onset of continuous symptoms until the surgery date (8 yr). Two of the 10 patients had MEN1, and both had a history of primary hyperparathyroidism; 1 additionally had a previous pituitary adenoma, and the other had a positive family history, all indicative of MEN1. Table 2Go shows the tumor-specific characteristics. All patients had elevated fasting serum gastrin levels, and the majority had elevated preoperative secretin-stimulated or calcium-stimulated gastrin release indicative of active Zollinger-Ellison syndrome (35). The location and extent of the tumor encountered during surgery were comparable to those in other series (4) in that the duodenum and the pancreas were the primary tumor sites in more than half of the patients, 40% included the primary site and metastases to lymph nodes, and 2 patients had evidence of liver metastases during surgery. During the postoperative follow-up (1–76 months; mean follow-up of 27.6 months), 4 patients remained disease free (patients 3, 4, 8, and 10; Table 2Go). Tumors from 2 patients displayed an aggressive growth pattern (patients 5 and 9; Table 2Go), and tumors in 4 patients did not demonstrate growth (Table 2Go).


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  		Table 1. Clinical characteristics of patients

 

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  		Table 2. Tumor characteristics in studied patients

 
When tumor RNA was extracted from frozen tissue from eight gastrinomas and analyzed by competitive PCR, the presence of transcripts for CaR could be identified in all tumors. When no RT was performed no product was obtained with the primers used, demonstrating that the primers did not amplify genomic DNA. Figure 1Go shows an example of the competitive PCR for the CaR from patient 1 with a primary gastrinoma of the heart and patient 2 with liver metastases. Where the intensity of the ethidium bromide staining of the upper band equals that of the lower band is the determined amount of molecules in the unknown sample. In these two examples the gastrinoma from patient 1 had a 16-fold greater amount of CaR than that from patient 2 when corrected for the variable input amount using ß-actin (Table 2Go). The results of CaR concentrations for all eight patients normalized to the amount of ß-actin present in the sample are shown in Table 2Go. The results indicate a wide spectrum of CaR mRNA amounts in the different tumors varying over an 80-fold range. The lowest value of 0.4 CaR/ß-actin molecules was in a duodenal gastrinoma (Table 2Go, patient 7), and the highest value of 3.16 CaR/ß-actin molecules was in a cardiac gastrinoma (Table 2Go, patient 1).



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  		Figure 1. Ethidium bromide staining of the competitive PCR for the CaR from the tumors of patients 1 and 2 (Table 2Go) in a 1% agarose gel. The arrow indicates where the amount of unknown equaled the amount of competitor and represented the amount of CaR in the unknown sample.

 
To verify the protein expression of the CaR in gastrinomas and obtain information about the molecular size, we performed a Western blot analysis of a pancreatic gastrinoma (patient 3, Table 2Go and Fig. 2Go). In lane 1 of Fig. 2Go, the cell line HEK-293 stably transfected with the native CaR served as a positive control and showed bands at 250 and 140 kDa as reported previously (36). The band at 250 kDa represents the dimer of the CaR, whereas the band at 140 kDa corresponds to the fully glycosylated monomer (37). In lane 2, showing a gastrinoma, there was the presence of a broad band centered at 250 kDa, which demonstrates a large range of molecular masses of the dimerized or aggregated form of the CaR in the tumor cells. There were also a 140-kDa band and a 120-kDa band, which probably represent the fully glycosylated monomeric form and possibly a slightly deglycosylated degradation product, respectively. Pretreatment with 100 mmol/L iodoacetamide and 50 mmol/L ß-mercaptoethanol led to a shift from the receptor dimer to the primarily lower molecular mass form in the gastrinoma (Fig. 2Go, lane 3).



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  		Figure 2. Western blot analysis of a metastatic pancreatic gastrinoma with the CaR-specific antibody (ADD) from patient 3 (Table 2Go). Equal amounts of total cellular protein were loaded in each lane. Lane 1 shows results of HEK-293 cells stably transfected with the wild-type CaR. Lanes 2 and 3 show samples from a single tumor, but differently pretreated, as indicated at the bottom. The positions of molecular weight markers are shown on the left margin.

 
Immunohistochemical staining with the same antibody (Figs. 3Go and 4Go) showed positive staining in virtually all cells in each of the three tumors tested. When nonimmune serum or no antibody was added, there was no staining (data not shown). Figure 3AGo shows the hematoxylin and eosin stain of a gastrinoma metastatic to the liver (Table 2Go, patient 9), with the adjacent section (Fig. 3BGo) stained for the CaR. The staining pattern of the CaR is specific for the tumor, with no staining of the normal liver. Figure 3CGo demonstrates that the staining is primarily membranous, although in cells, positive staining in the cytoplasm was also observed. Figure 3DGo shows CaR staining in a gastrinoma metastatic to the liver from a different patient (Table 2Go, patient 10) and indicates receptor expression in this tumor (compare Fig. 3Go, B and D). To assess the CaR staining pattern in normal duodenum and pancreas we performed immunohistochemical staining in specimens from patients with gastrinomas in these locations. The normal pancreas showed specific CaR staining in the islets (Fig. 4Go, top panel) with complete absence of staining in the exocrine pancreas. In the normal duodenum (Fig. 4Go, bottom panel) there was minimal CaR staining limited to a few endocrine cells in the crypts. The submucosal gastrinoma (from patient 6, Table 2Go,) showed dense CaR staining. Normal lymph nodes and liver were negative in all cases.



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  		Figure 3. Immunohistochemistry of the CaR in normal liver and liver metastases from gastrinomas in two patients. A, Hematoxylin-eosin stain of the normal liver and a liver metastasis at low power (patient 9, Table 2Go; x40). The adjacent slide was stained with the CaR-specific antibody ADD (B). The brown staining represents the CaR. In C, a higher power view (x100) of the same CaR-stained tumor is depicted to demonstrate the membranous staining pattern. D, A low power view of CaR staining of a liver metastasis from a different patient (patient 10, Table 2Go; x40), demonstrating the variable expression in two metastatic tumors (compare to B).

 


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  		Figure 4. Immunohistochemistry of the CaR in normal pancreas and duodenum as well as a duodenal gastrinoma. The upper panel shows the hematoxylin-eosin stain of the normal pancreas at low power (left; x40) and the adjacent slide stained with the CaR specific antibody ADD (right). The normal islets are shown to contain CaR, whereas normal acini are negative. In the lower panel (left), the hematoxylin-eosin stain in a low power view (patient 6, Table 2Go; x40) of the duodenum with a submucosal gastrinoma is shown. The lower panel (right) shows the CaR staining of the adjacent slide (x40).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study we provide several lines of evidence that the CaR is expressed in gastrinomas. First, the presence of mRNA of the CaR was detected in variable amounts in all tumors using RT-PCR amplification. That the amplified products actually represented the CaR cDNA was confirmed by sequencing. Furthermore, the primers were designed to span intron sequences, and no products were amplified using genomic DNA as PCR template or when the reverse transcriptase step was omitted. Moreover, PCR products amplified with similar primers have been used to examine the presence of this receptor in various tissues (breast, parathyroid) (30). Secondly, the monoclonal antibody used in this study (ADD) to demonstrate the presence of CaR protein is directed against amino acids 214–235 of the human CaR. It has been shown to be specific for detection of the human CaR in both human CaR-transfected cells (37) and human antral cells (38). Thirdly, the conclusion that the CaR is present in gastrinomas and not the surrounding normal tissue was supported by the immunohistochemical results. Immunohistochemistry demonstrated no detectable CaR in normal liver or acinar cells of the pancreas. However, CaR was present in a metastatic pancreatic gastrinoma to the liver and normal pancreatic islets. Furthermore, only the numerous deposits of tumor cells of a duodenal gastrinoma showed prominent staining for the CaR protein. Similarly, Western blot analysis with ADD of normal lymph nodes, liver, or colon tissue did not show the presence of the CaR.

Previous studies have shown that the CaR is expressed in various tissues. It was originally cloned from bovine parathyroid cells (22). Subsequently, the receptor has been localized to human parathyroid (39), kidney cells (40), parafollicular thyroid cells (C cells) (41), and intestinal epithelial cells (42). The finding of functional CaR in intestinal cell cultures is seemingly in contradiction to the lack of immunohistochemical staining in the duodenum in our study. However, one reason for this discrepancy could be the methods used. Using RT-PCR (2 rounds x 35 cycles) and Northern blot, Gama and collaborators (42) found expression of the CaR in cell cultures derived from colonic adenocarcinomas and in rat intestinal preparations. The level of expression needed for detection by immunohistochemistry as employed in our studies is generally higher than that obtained when using molecular amplification strategies. Therefore, CaR mRNA could be detected by PCR amplification, but the protein could be missed by immunohistochemistry. Western blot analysis of a gastrinoma demonstrated major bands at 250 and 140 kDa. Previous studies in cells transfected with the native CaR have shown that the band at 250 kDa corresponds to a disulfide-linked dimer of the CaR, and the band could be shifted to the monomeric CaR by using reducing conditions during SDS-PAGE (43). The band at 140 kDa represents the fully glycosylated monomer of the CaR, as demonstrated by the shift from 140 to 120 kDa after complete deglycosylation (44). Our studies indicate that the CaR is present in gastrinomas predominantly as the 250-kDa glycosylated receptor dimer. That this represented the dimer form was supported by the effects of reducing agents, which resulted in a proportional increase in the 140-kDa form of the CaR in a gastrinoma. Immunohistochemistry with the same monoclonal antibody (ADD) of the gastrinomas showed positive staining limited to the tumors and endocrine cells in the normal tissue, as seen in the duodenum and pancreas. The staining pattern in the tumors was mostly membranous, corresponding to binding of the antibody to the mature receptor in the plasma membrane. The degree of staining varied among the tumors, potentially reflecting the variable amounts of CaR mRNA measured in the tumors.

The function of the CaR in gastrinoma cells is unknown. In most tissues studied to date the primary role of the CaR is to control extracellular Ca2+ homeostasis. The control of PTH secretion from the parathyroid gland, reflective of the extracellular Ca2+ concentration, is thought to be mediated by the CaR (45). Similarly, the CaR in the kidney is thought to control Ca2+ excretion (45). Evidence for this was gathered from mutations in the CaR gene resulting in attenuated functional responses leading to neonatal severe hyperparathyroidism and familial hypocalciuric hypercalcemia (46). In the intestine the CaR has been proposed to have a role in controlling intestinal Ca2+ absorption (42). Besides a role in Ca2+ homeostasis, the CaR probably has other important roles in other tissues that are not yet clearly defined. The CaR has been isolated from the brain cells (47, 48), gastric cells (49), human breast tissue (30), and keratinocytes (50). In these differing cell populations the CaR has been proposed to have a variety of functions. In the rat hippocampus it may be involved in long-term potentiation, a putative in vitro analog of memory, and differentiation (47). The role of the CaR in the stomach has not yet been clearly defined. The presence of the CaR in antral G cells has provided yet another function for this receptor (38). This latter report included the observation that increasing Ca2+ concentrations led to increasing gastrin release from antral G cells and provides evidence for mediation of the secretagogue function of Ca2+ on endocrine cells. Moreover, the pharmacological compound KRN 568, which acts on the CaR, was able to increase serum gastrin levels in healthy volunteers (51). Furthermore, the CaR has been identified in human insulinoma cell cultures (21). It is known that insulinoma cells respond to increased extracellular Ca2+ concentrations with a release of insulin, and this effect has been used for clinical localization procedures (3, 12). Therefore, the presence of the CaR in gastrinomas may have several functions. As systemic extracellular Ca2+ application can elicit a measurable gastrin release in patients with gastrinoma, and we have demonstrated the presence of the CaR in gastrinomas, it can be proposed that this hormone release is at least partially mediated via the CaR. As calcium-stimulated gastrin release by the gastrinomas can vary over a 500-fold range, it is at present unknown whether the density of the CaR on the tumor is a factor in determining the magnitude of the hypergastrinemia. Furthermore, the CaR may mediate growth or differentiation signals in gastrinomas, as seen in other malignancies. In keratinocytes and fibroblasts the CaR has been hypothesized to partially regulate differentiation and proliferation (50, 52). This growth-regulating function of the CaR is corroborated by the responsiveness of cell cultures from human colonic neoplasms to extracellular Ca2+ concentrations (53) and the presence of the CaR in such cells (42). Furthermore, the CaR has been shown to stimulate the growth of various cell lines (54, 55, 56) and oligodendrocytes (47) and when transfected into cells that normally do not express the CaR (57). Recent studies show that a proportion of gastrinomas have an aggressive growth pattern (26, 58), and the factors that govern this growth pattern are largely unknown. In the present study the amount of the CaR in different gastrinomas varied over an 80-fold range. Whether the CaR has an important role in growth or differentiation of gastrinomas, as seen in other cells (50, 52, 54, 55, 56, 59), remains to be determined.

The Ca2+-evoked release of gastrin from the gastrinoma tissue may not be solely mediated by the CaR. This conclusion is supported by the observation that in antral G cells as well as pancreatic ß-cells activation of voltage-gated calcium channels (L type) stimulate hormone release (60, 61). We did not examine the presence or role of voltage-gated calcium channels in gastrinomas, but it is conceivable that activation of these channels could also influence the gastrin response to Ca2+ administration.

In summary, we found that the CaR is universally expressed in gastrinomas regardless of whether a genetic predisposition (MEN1) was present. Furthermore, the receptor is expressed in varying amounts in the different tumors. The proposed functional role of this receptor is the mediation of Ca2+-stimulated gastrin release from the tumors. Further studies are necessary to characterize the exact contribution of this receptor to the hormone release, potential other functions, and possible clinical utility of its detection.

Received November 10, 1999.

Revised July 31, 2000.

Accepted August 7, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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