Scandinavian Journal of Gastroenterology
ISSN: 0036-5521 (Print) 1502-7708 (Online) Journal homepage: http://www.tandfonline.com/loi/igas20
Session 3: The Gastrin Receptor Assay P. M. Kleveland & H. L. Waldum To cite this article: P. M. Kleveland & H. L. Waldum (1991) Session 3: The Gastrin Receptor Assay, Scandinavian Journal of Gastroenterology, 26:sup180, 62-69, DOI: 10.3109/00365529109093180 To link to this article: http://dx.doi.org/10.3109/00365529109093180
Published online: 08 Jul 2009.
Submit your article to this journal
Article views: 4
View related articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=igas20 Download by: [University of California, San Diego]
Date: 22 March 2016, At: 16:33
The Gastrin Receptor Assay P. M . KLEVELAND & H. L. WALDUM Dept. of Medicine. University Hospital. 'Trondheim, Norway
Downloaded by [University of California, San Diego] at 16:33 22 March 2016
Kleveland PM, Waldum HL. The gastrin receptor assay. Scand J Gastroenterol 1991, SUPPI PI 180), 62-69 Gastrin is a major physiologic regulator of gastric acid secretion and growth of the oxyntic mucosa. Biologically active radiolabelled hormones may be used to characterize and localize receptors for peptide hormones. The cellular localization of the gastrin receptor in the fundic mucosa, however, is still a matter of great debate owing to difficulties in developing a gastrin receptor binding assay. Despite considerable work in several laboratories. the criteria for true receptor binding have not yet been fulfilled. The preparation of a suitable tissue receptor material (plasma membranes o r isolated cells) from the heterocellular fundic mucosa seems to be the major problem. This problem may be related to the fact that the receptors are only present on the enterochromaffin-like cells (ECL). which constitute but a minor fraction of the cells in the oxyntic mucosa. Furthermore, the second messenger of gastrin is still not known, and the poor functional responsiveness of isolated cells and the oxyntic glands to gastrin further complicates the evaluation of the gastrin receptor. In this review the different steps in the gastrin receptor assay (the labelling of gastrin, preparation of the receptor, and the incubation and correlation of the binding and biologic effect of gastrin) are discussed. Key words: -4ssay; binding; gastrin: receptor Per M . Kleveland, M . D . , Dept. of Medicine, University Hospital, N-7006 Trondheim, Norway
The main physiologic actions of gastrin are the stimulation of gastric acid secretion and growth of the oxyntic mucosa (1). Since Gregory &Tracy (2) and Gregory et al. (3) isolated, purified, and determined its amino acid sequence. gastrin has had an accepted and established role in the physiologic regulation of gastric acid secretion. As with other regulatory peptides, gastrin exerts its biologic effects by binding to a specific receptor on the plasma membrane of the target cell. The binding activates membrane enzymes, leading to the liberation of an intracellular second messenger. The binding of biologically active radiolabelled regulatory peptides provides a powerful tool for characterization and localization of receptors (4). The initial binding of the labelled peptide to the receptor seems to proceed as a bimolecular reaction in accordance with the law of mass ( 5 ) . The binding data from equilibrium conditions may be used for the characterization of the
binding reaction and, indirectly, the binding site. Thus the number of binding sites and the dissociation constant ( K d ) may be calculated by a Scatchard transformation of the dose response curve (6). To represent receptor binding, the calculated Kd should be similar t o the concentration of non-labelled hormone displacing 50% of the labelled hormone (CD,,,) (7). Additionally, for a hormone, the K d and CDS(, should be compatible with the physiologic plasma concentration. Furthermore, the binding should be saturable, indicating a finite binding capacity or number of receptors. Third, the binding should be specific for the hormone, and binding affinities of analogues should reflect their biologic activity. Moreover, the binding should be restricted to organs on which the peptide has an effect. Preferably, the binding should be correlated with a biologic response (8). In 1976 Lewin and co-workers (9, 10) were the first t o demonstrate specific binding of a gastrin
Downloaded by [University of California, San Diego] at 16:33 22 March 2016
Gastrin Receptor Assay
label to rat gastric fundic mucosa. They used tritiated gastrin with a low specific radioactivity (60 Ci/mmol). Binding sites were described both on plasma membranes and on isolated cells, but the affinity (&; 9 nM) was not in the physiologic range. Three years later, Takeuchi et al. (11) described their gastrin receptor assay, using a crude plasma membrane fraction from rat fundic mucosa and iodinated gastrin with a high specific radioactivity as a tracer. High-affinity binding sites (Kd; 0 4 nM) were detected, and the gastrin receptor in the oxyntic mucosa was biochemically characterized (12). In 1984 Sol1 et al. (13) described specific high-affinity binding of '251-15leucine gastrin to dispersed canine fundic mucosal cells (13). Using elutriation to separate these cells by size, they were able to show that the binding correlated with the parietal cell content. Several laboratories have since reported modifications of the gastrin receptor binding assay. Different ligands and preparations of fundic mucosa have been used (Table I). Highaffinity binding sites have been demonstrated in the oxyntic mucosa in different species, including the rat (14), dog (13), rabbit (15), pig (16), and guinea-pig (17). Similar binding sites have been found in colon cancers (14,18), in some colon (14, 19) and stomach (20,21) cancers, but only inconstantly in duodenum (18). However, functional studies indicate that in the rat (22) and even in other species (23,24), the acid-stimulatory effect of gastrin may be
mediated by liberated histamine. In the rat histamine is produced (25) and released from the enterochromaffin-like (ECL) cell. In other species, too, the E C L cell produces histamine (26), and may be the source of the histamine participating in the stimulation of the acid secretion (23,24). It should be also recalled that gastrin has a specific trophic effect on the ECL cell in most species (27-30). Therefore, there must be a gastrin receptor on the E C L cell, and functionally there is no need for a gastrin receptor on the parietal cell (24). If the gastrin receptor is located only on the E C L cell (24), representing only a few per cent of the cells in the oxyntic mucosa (31,32), it is easily imagined that the receptor concentration may be too low to enable detection in binding experiments. In fact, this could be the explanation of the extraordinary difficulties in establishing a reproducible and accepted gastrin receptor assay. The principle of the gastrin receptor binding assay is the use of a biologically active labelled gastrin, which is incubated in vitro with a tissue receptor preparation. After the incubation period the hormone bound to the tissue is separated from the unreacted label by either centrifugation or filtration, and the bound radioactivity is counted. The development of a gastrin receptor binding assay, accordingly, involves several critical steps to be evaluated: the production of a stable gastrin tracer with preserved biologic activity, the preparation of a suitable tissue
Table 1. Biochemical characteristics of the gastrin receptor Ligand '251-G1-17 1ZSI-'51eu-G1-17t IZsI-'Me-G1- 17$ 1ZsI-'51eu-G2-17 '251-'51e~-G217
* Kd = dissociation constant. t Leu = leucine.
t
Nle = norleucine.
63
Fundic tissue preparation Plasma membrane fraction Isolated cells Isolated cells Isolated glands Detergent solubilized gastric mucosa
Kd* 0.4-1.1 0.46 0.07 2.3 300
Species reference Rat (14, 55)
Dog (13) Rabbit (15) Guinea-pig (17) Pig (16)
64
P. M . Kleueland & H . L. Waldum
preparation from the oxyntic mucosa, the conditions during incubation, and the correlation of binding to the biologic response of gastrin.
Downloaded by [University of California, San Diego] at 16:33 22 March 2016
THE LABELLED GASTRIN Gastrin circulates in plasma at a concentration in the lower pM range. The high affinity between gastrin and its receptor makes it necessary to produce labelled gastrin with high specific activity for use in binding studies. The radioactivity of labelled gastrin with a single '''1 molecule would be greater than for single substitutions with either "1 or "C by a factor of 72 and 32,000, respectively (4). Iz5I is accordingly the most suitable radionucleotide for producing labelled gastrin for receptor-binding studies. Highly enriched isotope preparations with a nearly 100% abundance of '?I (2150 Ci/mmol) are now available, and it is thus possible to produce monoiodinated tracers with about the same specific radioactivity (13. 15,33). The concentration of the tracer is determined by radioimmunoassay after identical immunoreactivity of the tracer is confirmed by comparison with unlabelled gastrin (that is, they have superimposable binding curves). The radioactive gastrin, however, may lose biologic activity as a result of the labelling process (34) or by the mere presence of the I atom itself (35). Data concerning biologic activity of the radioiodinated gastrins used for receptor binding are therefore of great importance. The assessment of biologic activity of labelled gastrin, however, has been cumbersome and difficult. The usual in vivo bioassays for gastrin have several disadvantages: they are laborious, lack precision, may be influenced by metabolism and excresion, and require large amounts of label (36). Therefore, macroscale iodinations (mixtures of '"I and '?'I) producing gastrin of low specific radioactivity have been performed to test biologic activity of the iodinated gastrin (37). The tracer itself, with high specific activity exposed to radioactivity during iodination and storage and used for receptor binding studies, was not tested (11, 14). The bioassay of gastrin. however, was recently significantly improved by the development of the totally isolated vascularly
perfused rat stomach (38,39). This bioassay is sensitive to gastrin in physiologic concentrations, requires only small amounts of gastrin, and was found useful for direct testing of the routine laboratory tracers with high specific activity (33). Quite recently, this model has been further improved by determination of the immediate histamine release into the venous drainage (40). Chloramine-T oxidation has been the method most widely used for iodination of gastrin (41). However, it may destroy the molecule, leading to loss of biologic activity (34). Iodination performed even with short reaction times and at low chloramine-T/gastrin molar ratios may give biologically inactive gastrin tracers (Kleveland and Waldum, unpublished observations) (Fig. 1). Chloramine-T oxidizes methionine and probably also the tryptophan residue, resulting in a total loss of biologic activity (34, 42,43). This is somewhat at odds with earlier studies, which reported that ultrashort chloramine-T oxidation does preserve the biologic activity of the gastrin tracer (37). Solid-phase iodination with Iodogen may give less damage to the gastrin molecule and thus retain biologic activity (44). Tracers prepared with Iodogen have been demonstrated to retain biologic gastrin activity in gastric fistula rats (36), dogs (14), and in the totally isolated vascularly perfused rat stomach (33) (Fig. 1). Nevertheless, Iodogen may also oxidize methionine, but probably only to its sulphoxide, which is biologically active (43). This could explain the minor difference in the biologic activity of iodinated gastrin prepared by chloramine-T and that prepared by Iodogen iodinations. To prevent the oxidation of methionine, analogues using leucine o r norleucine substitutions for 15-methionine have been synthesized (45,46). These gastrin analogues are reported to have full biologic activity and to be more resistant to oxidation (11,13,37). They seem to be an attractive alternative when preparing gastrin tracers for receptor binding studies. Moreover, 15-norleucine gastrin is chemically the closest to S m e t h i o n i n e gastrin and therefore theoretically the most ideal analogue (43). Whereas 1251-gastriniodinated by a chloramineT method tends to be rather unstable and give
Gastrin Receptor Assay
G
IMX
1-17
I I I I I
T
I
A
I I
Downloaded by [University of California, San Diego] at 16:33 22 March 2016
65
T
----.-p-.-....p. .+....... .....I.
I
Y
"2 0
40
I
60
.....I.
1
, a l p ]
80
100
TIME ( m i d Fig. 1. A comparison between the biological effect of G1-17 (0----0). "'I-G 117 (Iodogen) ( 0 - 0 1 , and '251-G1-17(chloramine-T) (W-W) at a concentration of 65 pM on the acid secretion by the totally isolated vascularly perfused rat stomach co-stimulated with isobutylmethylxanthine at a concentration of 50 pM. For comparison the base-line ( A---- A ) and maximal gastrin stimulated acid secretion (0----U) are given (Ref. 33). Chloramine-T iodination was performed as described in Ref. 37.
low specific binding (13), 1251-gastrinprepared by Iodogen is reported to give reproducible high specificbinding (14). Iodinated 15-leucine (11, 13) and 15-norleucine analogues (15) also give biologically active gastrin tracers suitable for receptor studies. Similar Kd and number of binding sites on isolated canine parietal cells have been reported with iodinated gastrin and gastrin analogues prepared by the Iodogen method (43). RECEPTOR PREPARATIONS Gastrin receptors have mainly been studied in a plasma membrane fraction from homogenized oxyntic mucosa or on dispersed cells and, recently, o n gastric glands from the oxyntic mucosa. PLASMA MEMBRANE FRACTIONS Plasma membrane fractions are easy to prepare
and can be stored and used for screening
for receptors in most organs. By different centrifugation techniques combined with enzyme markers, plasma membranes of various degrees of purification from oxyntic mucosal homogenates have been prepared (9,11,14). However, there have been some major problems. The homogenization and purification steps are often time-consuming and rather crude mechanical procedures that expose the receptors to liberated proteolytic enzymes. Retained proteolytic activity in the final receptor preparation can affect both the receptor and the labelled gastrin during incubation (10). The main problem, however, seems to be the heterogeneity of the cells in the oxyntic mucosa. Plasma membranes from dispersed fundic mucosal cells enriched with a particular cell type have not, to our knowledge, been successfully used. Since the cells possessing the gastrin receptor may only constitute a small
Downloaded by [University of California, San Diego] at 16:33 22 March 2016
66
P. M. Kleuelund & H . L. Wuldum
fraction of the total fundic mucosal tissue, the non-specific binding will tend to be too high to enable detection of the specific billding. However, in 1979 a crude plasma membrane fraction from fundic mucosa was reported suitable for gastrin receptor binding by Takeuchi et al. ( 1 I ) . The gastrin receptor was extensively studied and biochemically characterized (12). The method has, however, never been reproduced by other laboratories. Several significantly different modifications of the assay have been reported to detect specific binding (14, 18). The poor reproducibility and inconsistency of the results make the method rather uncertain. DISPERSED CELLS A N D GLANDS Isolated fundic mucosal cells are easy to prepare and offer the advantage that the cell mixture can be fractionated in accordance with the cell type of interest (13). It is not possible to assess acid secretion directly from isolated parietal cells, but it may be done indirectly by the incorporation of the weak base aminopyrine (47). Unfortunately, gastrin has been found to be a weak and inconstant stimulator of the aminopyrine uptake in isolated parietal cells (48) and isolated gastric glands (47). Moreover. the biologic activity of gastrin on the cellular level has been difficult to assess. since its second messenger has not yet been established (49). The treatment of the tissue with pronase and/or collagenase may alter the biologic function of the receptors (4). Pronase has a more general proteolytic activity than collagenase and may be expected to damage the receptor more easily. To isolate cells from a thick-walled stomach like the rat stomach. i t is necessary to use pronase (SO). Nevertheless, parietal cells prepared by the use o f either collagenase and/or pronase in addition to ethylenediaminetetraacetic acid (EDTA) to bind Ca*+ have retained responsiveness towards histamine and carbacholine (51). Gastrin. however. has no or only faint stimulatory effect o n the aminopyrine uptake in isolated parietal cells and gastric glands (47, 48. 51). Sol1 et al. (13). using isolated canine parietal cells enriched by elutriation. have confirmed some of the binding data obtained in the rat plasma membrane
fraction. They used chloramine-T-labelled 1 Sleucine gastrin and correlated the specific binding with the accumulation of aminopyrine in parietal cells. Binding of gastrin, however. was also detected in fractions containing smaller cells, and the parietal cell fraction used was not completely pure (13). Isolated gastric oxyntic glands have recently been reported to possess high-affinity binding sites for gastrin (17), but this method does not establish the cellular localization of the receptor. Gastrin-binding proteins have quite recently been identified in detergent extracts from whole gastric fundic mucosa (16), isolated parietal cells (52). and gastric glands (17) by covalent crosslinking of iodinated G2.17 to elucidate the molecular structure of the gastrin receptor. The gastrin proteins, however, had very low affinities for the gastrin probe (16). INCUBATION The specific binding has been estimated as the difference between binding without (total binding) and with a thousandfold excess of unlabelled gastrin (non-specific binding). A5 for other peptide hormones incubation damage is a major factor to be avoided when establishing stable binding to a tissue preparation (53). Incubation damage can potentially affect both label and receptor. It is usually minimal when using whole intact cells and greatest with plasma membranes from whole homogenates (53). Accordingly, Soil et al. (13) reported isolated canine cells to degrade only 11% of their chloramine-T-iodinated '51eucine-G,.,,, and Ramani et al. (17) reported a 25% degradation of an identical but less purified label with isolated guinea-pig gastric glands after 30 min at 37°C. In the latter study the activity was assumed to be proteolytic, but protease inhibitors were reported to be without any effect (17). Substantial incubation damage has recently been demonstrated in the standard crude 270- to 30,000-g crude plasma membrane fraction from fundic mucosal homogenates (18). The original preincubation and incubation conditions at 30°C. introduced by Brown & Gallagher (54) and later
Downloaded by [University of California, San Diego] at 16:33 22 March 2016
Gastrin Receptor Assay
adopted by Takeuchi et al. (11) were later shown to give variable and mainly non-specific binding (36). Althoughthe degradation was demonstrated to be partially proteolytic, protease inhibitors were ineffective (18). The damage to the label and the binding site was only controlled by reducing the incubation temperature to 15"Cand omitting the preincubation. Thus, reproducible specific binding t o fundic plasma membranes with a Kd of 0.8 nM (Scatchard analysis) was detected (18). This variant of the assay was later reproduced by Rossowski et al. (55). Singh et al. (14), also by omitting the preincubation period and by repetitive washing of the fundic mucosal homogenate, have recently reported high-affinity binding to fundic mucosa at 30°C, using Iz5I-gastrin iodinated with the help of Iodogen. Controls for incubation damage, however, were not presented. Damage to IZ5Igastrin with plasma membranes from fundic homogenates at 30°C was noticed already in the original assay presented by Brown & Gallagher (54). Controls for potential damage were not included in the studies of Takeuchi et al. ( l l ) , who adopted this method for characterization of the gastrin receptor in rat fundic mucosa (12). Damage to the label and loss of receptor activity may seriously affect calculations of binding affinities (&) and the number of binding sites. CORRELATION BETWEEN BINDING A N D BIOLOGIC EFFECT The direct measurement of acid is only possible in intact fundic mucosa. Any correlation between the binding of labelled gastrin to plasma membranes and a biologic effect it has so far not been possible to evaluate. Specific binding to rat fundic plasma membranes was recently indirectly shown to correlate with biologic activities of the label in the totally isolated vascularly perfused rat stomach during storage of an Iodogen-labelled '"I-gastrin (56). This study indicates that the specific binding does reflect an interaction with the receptor (56). Other criteria for true receptor binding, however, were not satisfactorily fulfilled, making the interpretation difficult. The strongest evidence for a direct correlation
67
between specific binding and the biologic effect of gastrin has been reported on isolated canine fundic mucosal cells (13). Specific binding of a '251-labelled 15-leucine gastrin analogue prepared with chloramine-T was shown to be correlated with the accumulation of aminopyrine when the '*'I label of the gastrin analogue was used as a stimulant (13). Although some specific binding was observed in a fraction with small cells, the binding was mainly confined to fractions containing the parietal cells (13). The authors and many others have considered this study to be major proof of the existence of a functional gastrin receptor on the parietal cell, at least in the dog. The data, however, are still discussed in view of the inconsistent and weak effect of gastrin and the lack of differential counting and characterization of the cells present in the parietal cell fraction used. The stimulatory effect could therefore possibly be accounted for by coelution of parietal and histamine-producing cells. Histamine-producing cells have recently been reported to exist in the dog fundic mucosa as well (57). Nevertheless, quite recently, monoclonal antibodies have been produced by immunization with canine parietal cell fractions and postulated to be directed towards a gastrin receptor on the parietal cell (58). The monoclonal antibodies inhibited the binding of labelled gastrin and reduced aminopyrine uptake in the isolated canine parietal cells used (58). CONCLUSION The gastrin receptor binding assay apparently still does not fulfil the criteria for true receptor binding. Both the cell type possessing the receptor and its second messenger are still under discussion. A new non-peptidic gastrin antagonist (59) will probably be a useful tool to characterize and localize the gastrin receptor further.
REFERENCES 1. Walsh J H , Grossmann MI. Medical progress:
gastrin. N Engl J Med 1975, 292, 13241334 2. Gregory RA, Tracy HJ. The preparation and properties of gastrin. J Physiol (Lond) 1961, 156, 523-543
Downloaded by [University of California, San Diego] at 16:33 22 March 2016
68
P. M . Kleveland & H. L. Waldum
3. Gregory RA, Hardy PM, Kenner GW, Sheppard RC. Structure of gastrin. Nature 1964, 204, 931933 4. Gardner JD. Receptors for gastrointestinal hormones. Gastroenterology 1979, 76, 202-214 3 . Catt KJ, Dufau ML. Introduction: The clinical significance of peptide hormone receptors. Clin Endocrinol Metabol 1983, 12, XI-XLIV 6.Scatchard G . The attractions of proteins for small molecules and ions. Ann NY Acad Sci 1949. 51, 66&672 7. Akara T, Cheng V-JK. A simple method for the determination of affinity and binding site concentration in receptor binding studies. Biochim Biophys Acta 1977. 470, 412-423 8. Lin S-Y, Goodfriend TL. Angiotensin receptors. Am J Physiol 1970. 218. 1319-1328 9. Lewin M. Soumarmon A, Bali JP, et al. Interaction of %labelled synthetic human gastrin I with rat gastric plasma membranes. Evidence for the existence of biologically reactive gastrin receptor sites. FEBS Letters 1976, 66. 168-172 10. Soumarmon A. Cheret AM, Lewin MJM. Localization of gastrin receptors in intact isolated separated rat fundic cells. Gastroenterology 1977. 73. 90tL903 1 1 . Takeuchi K. Speir GR, Johnson LR. Mucosal gastrin receptor. I. Assay standardization and fulfillment of receptor criteria. Am J Physiol 1979. 237. E284E29.1 12. Johnson LR. Gastrin receptor assay. Meth Enzymol 1985. 109, 56-64 13. Sol1 AH, Amirian DA, Thomas LP. Reedy TJ, Elashoff JD. Gastrin receptors on isolated canine parietal cells. J Clin Invest 1984, 73. 1434-1447 1.2. Singh P, Rae-Venter B, Townsend CM Jr. Khalil T. Thompson JC. Gastrin receptors in normal and malignant gastrointestinal mucosa: age-associated changes. Am J Physiol 1985. 249, G761-769 IS. Magous R. Bali J-P. High affinity binding sites for gastrin on isolated rabhit gastric mucosal cells. Eur j Pharmacol 1982. 82. 17-54 16. Baldwin GS. Chandler R, Scanlon DB, Weinstock J . Identification of a gastrin binding protein in porcine gastric mucosal membranes by covalent cross-linking with iodinated gastrin 2-17. J Biol Chem 1986. 261. 12252-12257 17 Kainani N, Praissman M. Molecular identification and characterization of the gastrin receptor in guinea pig gastric glands. Endocrinology 1989. 124. 1881-1887 I X Klrveland PM. Waldum HL. Binding and degradation of "'I-gastrin by plasma membranes from homogenized rat gastric mucosa. %and J Gastroenterol 1986. 21. 547-555 19 Upp JR Jr. Singh P, Townsend CM, Thompson JC. Clinical significance of gastrin receptors in human gastric colon cancers. Cancer Res 1989. 49. 48% 492 20 Kumamoto T. Gastrin receptors in human gastric scirrhous carcinoma. Gastroenterol Jpn 1988. 23. 384-389 21 Wein\tock J. Balwin GS Binding of gastrin,, to
human gastric carcinoma cell lines. Cancer Res 1989, 48, 932-937 22. Sandvik AK, Waldum HL, Kleveland PM, Schulze S ~ g n e nB. Gastrin produces an immediate and dose-dependent histamine release preceding acid secretion in the rat. Scand J Gastroenterol 1987, 22, 803-808 23. Waldum HL, Sandvik AK, Brenna E , Petersen H. The gastrin-histamine sequence in the regulation of gastric acid secretion. Gut 1990 (in press) 24. Waldum HL, Sandvik AK. Histamine and the stomach. Scand J Gastroenterol 1989, 24, 13&139 25. Hikanson R, Owman C. Argyrophilic reaction of histamine-containing epithelial cells in murine gastric mucosa. Experientia ,1969, 25, 626 26. HikansonR,BottcherG, EkbladE.etal. Histamine in endocrine cells in the stomach. Histochemistry 1986, 86, 5-17 27. Larsson H, Carlsson E, Mattsson. et al. Plasma gastrin and gastric enterochromaffin-like cell activation and proliferation. Gastroenterology 1986. 90, 391-399 28. Tielemans Y , Axelson J. Sundler F, Willems G, Hikanson R . Serum gastrin concentration affects the self replication rate of the enterochromaffinlike cells in the rat stomach. Gut 1990. 31, 274-278 29. Borch K, Renvall H, Liedberg G . Gastric endocrine cell hyperplasia and carcinoid tumors in pernicious anemia. Gastroenterology 1985, 88. 638-648 30. Bordi C, Coconi G , Togni R. Vezzadini P, Missale G. Gastric endocrine cell proliferation. Association with Zollinger-Ellison syndrome. Arch Patholl974, 98. 274278 31. Hikanson R , Larsson LI, Liedberg G. Oscarson J , Sundler F, Vang J. Effects of antrectomy or portacaval shunting on the histamine-storing endocrinelike cells in oxyntic mucosa of rat stomach. A fluorescence histochemical, electron microscopic and chemical study. J Physiol 1976, 259, 785-800 32. Rubin W, Schwartz B. Electron microscopic radiographic identification of the ECL cell as the histamine-synthesizing endocrine cell in the rat stomach. Gastroenterology 1979. 77, 458-467 33. Kleveland PM, Waldum HL, Bjerve KS. F j ~ s n e HE. Bioassay of gastrin using the totally isolated vascularly perfused rat stomach. A biomodel sensitive to gastrin in physiological concentrations. Scand J Gastroenterol 1987. 21, 945-950 34. Stagg BH, Temperley JM, Rochman H, Morley JS. Biological activity of iodinated gastrin. Nature 1970. 228, 58-59. 35. Kahn RC. Membrane receptors for polypeptide hormones. Meth Membr Biol 1975, 3. 81-146 36. Kleveland PM. Haugen SE, Waldum HL. The preparation of bioactive '"I-gastrin, using lodogen as oxidizing agent, and the use of this tracer in receptor studies. Scand J Gastroenterol 1985. 20, 569-576 37. Dockray GH. Walsh JH, Grossman MI. Biological activity of iodinated gastrins. Biochem Biophys Res Commun 1976. 69. 339-345 38. Kleveland PM. Haugen SE. Sandvik AK. Waldrum HL The effect ofu pentagastrin on the gastric
Downloaded by [University of California, San Diego] at 16:33 22 March 2016
Gastrin Receptor Assay secretion by the totally isolated vascularly perfused rat stomach. Scand J Gastroenterol 1986, 21, 379384 39. Kleveland PM, Haugen SE, Waldum HL. Effect of pentagastrin on the gastric acid secretion in the totally isolated vascularly perfused rat stomach stimulated with the phosphodiesterase inhibitor isobutyl methylxanthine. Scand J Gastroenterol 1986, 21, 577-584 40. Sandvik AK, Waldum HL. Rat gastric histamine release: a sensitive gastrin bioassay. Life Sci 1990, 46, 453-459 41. Stadil F, Rehfeld JF. Preparation of '251-labelled synthetic gastrin human gastrin I for radioimmunoanalysis. Scand J Clin Lab Invest 1972, 30, 361-368 42. Mourier G , Moroder L, Previero A. Prevention of tryptophan oxidation under iodination of tyrosyl residues in peptides. Z Naturforsch 1984,39b, 101104 43. Seet L, Fabri L, Nice EC, Baldwin GS. Comparison of iodinated (NleIs)- and (Met ")-gastrin,, prepared by reversed-phase HPLC. Biomed Chromat 1987, 2, 159-163 44. Fraker PJ, Speck JC Jr. Protein and cell membrane iodination with a sparingly soluble chloromide 1,3,4,6-tetrachloro-3a,6a-diphenylglycoluril.Biochem Biophys Res Commun 1978, 80, 849-857 45. Wunsch E , Deimer KH. Zur Synthese von HumanLittle-Gastrin-I und dessen Leucin-15 analogue. Hoppe-Seylers Z Physiol Chem 1972, 353, 12551258 46. Moroder L, Gohring W, Nyfeler R, Scharf R, Thamm P, Wendelberger G. Zur Synthese von Human-Little-Gastrin-I und dessen Leucin-15-, Norleucin- und Methionin-15-Analoga. HoppeSeylers Z Physiol Chem 1983, 3364, 157-171 47. Berglindh T , Helander HF, Obrink KJ. Effect of secretagogues on oxygen consumption, aminopyrine accumulation and morphology in isolated gastric glands. Acta Physiol Scand 1976, 97, 401-414 48. Sol1 AH. Secretagogue stimulation of "C-aminopyrine accumulation by isolated canine parital cells. Am J Physiol 1980, 238, G366G375
69
49. Morriset J. Gastrointestinal hormone receptors in the gut: localization, characterization, modulation, and ontogeny. In: Lebenthal E , ed. Human gastrointestinal development. Raven Press, New York, 1989, 99-122 50. Lewin M, Cheret AM, Soumarmon A , Girodet J. Methode pour I'isolement et la tri cellules de la muoqueuse fundique de rat. Biol Gastroenterol (Paris) 1974, 7, 139-144 51. Dial E, Thompson WJ, Rosenfeld GC. Isolated parietal cells: histamine response and pharmacology. J Pharmacol Exp Ther 1981, 219-585-590 52. Matsumoto M, Park J, Yamada T. Gastrin receptor characterization; affinity crosslinking of the gastrin receptor on canine gastric parietal cells. Am J Physiol 1987, 252, G142-G147 53. Ryan RJ, Lee CY. The role of membrane bound receptors. Biol Reprod 1976, 14, 1 6 2 9 54. Brown J, Gallagher ND. A specific gastrin receptor site in the rat stomach. Biochem Biophys Acta 1978, 538,42-49 55. Rossowski WJ, Ozden A, Ertan A, Maumus M, Arimura A. Somatostatin, gastrin, and cholinergic muscarinic binding sites in rat gastric, duodenal, and jejunal mucosa. Scand J Gastroenterol 1988, 23, 717-725 56. Kleveland PM, Waldum HL. The storage time of monoiodinated gastrin affects the biological activity and binding to rat fundic plasma membranes similarly, whereas the immunoreactivity is less affected. Scand J Gastroenterol 1987, 22, 390-396 57. Chuang CN, Toomey M, Chen MC, Sol1 AH. Histamine stores in the canine oxyntic mucosa: differentiation of two cell populations. Gastroenterology 1990, 98, A31 58. Mu F-T, Baldwin G , Weinstock J , Stockman D, Toh BH. Monoclonal antibody to the gastrin receptor on parietal cells. Proc Natl Acad Sci USA 1987, 84, 2698-2702 59. Huang SC, Zhang L, Chiang H-C, et al. Benzodiazepine analogues L365.260 and 364,718 as gastrin and pancreatic CCK receptor antagonists. Am J Physiol 1989, 257, G169-G174
ISSN: 0036-5521 (Print) 1502-7708 (Online) Journal homepage: http://www.tandfonline.com/loi/igas20
Session 3: The Gastrin Receptor Assay P. M. Kleveland & H. L. Waldum To cite this article: P. M. Kleveland & H. L. Waldum (1991) Session 3: The Gastrin Receptor Assay, Scandinavian Journal of Gastroenterology, 26:sup180, 62-69, DOI: 10.3109/00365529109093180 To link to this article: http://dx.doi.org/10.3109/00365529109093180
Published online: 08 Jul 2009.
Submit your article to this journal
Article views: 4
View related articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=igas20 Download by: [University of California, San Diego]
Date: 22 March 2016, At: 16:33
The Gastrin Receptor Assay P. M . KLEVELAND & H. L. WALDUM Dept. of Medicine. University Hospital. 'Trondheim, Norway
Downloaded by [University of California, San Diego] at 16:33 22 March 2016
Kleveland PM, Waldum HL. The gastrin receptor assay. Scand J Gastroenterol 1991, SUPPI PI 180), 62-69 Gastrin is a major physiologic regulator of gastric acid secretion and growth of the oxyntic mucosa. Biologically active radiolabelled hormones may be used to characterize and localize receptors for peptide hormones. The cellular localization of the gastrin receptor in the fundic mucosa, however, is still a matter of great debate owing to difficulties in developing a gastrin receptor binding assay. Despite considerable work in several laboratories. the criteria for true receptor binding have not yet been fulfilled. The preparation of a suitable tissue receptor material (plasma membranes o r isolated cells) from the heterocellular fundic mucosa seems to be the major problem. This problem may be related to the fact that the receptors are only present on the enterochromaffin-like cells (ECL). which constitute but a minor fraction of the cells in the oxyntic mucosa. Furthermore, the second messenger of gastrin is still not known, and the poor functional responsiveness of isolated cells and the oxyntic glands to gastrin further complicates the evaluation of the gastrin receptor. In this review the different steps in the gastrin receptor assay (the labelling of gastrin, preparation of the receptor, and the incubation and correlation of the binding and biologic effect of gastrin) are discussed. Key words: -4ssay; binding; gastrin: receptor Per M . Kleveland, M . D . , Dept. of Medicine, University Hospital, N-7006 Trondheim, Norway
The main physiologic actions of gastrin are the stimulation of gastric acid secretion and growth of the oxyntic mucosa (1). Since Gregory &Tracy (2) and Gregory et al. (3) isolated, purified, and determined its amino acid sequence. gastrin has had an accepted and established role in the physiologic regulation of gastric acid secretion. As with other regulatory peptides, gastrin exerts its biologic effects by binding to a specific receptor on the plasma membrane of the target cell. The binding activates membrane enzymes, leading to the liberation of an intracellular second messenger. The binding of biologically active radiolabelled regulatory peptides provides a powerful tool for characterization and localization of receptors (4). The initial binding of the labelled peptide to the receptor seems to proceed as a bimolecular reaction in accordance with the law of mass ( 5 ) . The binding data from equilibrium conditions may be used for the characterization of the
binding reaction and, indirectly, the binding site. Thus the number of binding sites and the dissociation constant ( K d ) may be calculated by a Scatchard transformation of the dose response curve (6). To represent receptor binding, the calculated Kd should be similar t o the concentration of non-labelled hormone displacing 50% of the labelled hormone (CD,,,) (7). Additionally, for a hormone, the K d and CDS(, should be compatible with the physiologic plasma concentration. Furthermore, the binding should be saturable, indicating a finite binding capacity or number of receptors. Third, the binding should be specific for the hormone, and binding affinities of analogues should reflect their biologic activity. Moreover, the binding should be restricted to organs on which the peptide has an effect. Preferably, the binding should be correlated with a biologic response (8). In 1976 Lewin and co-workers (9, 10) were the first t o demonstrate specific binding of a gastrin
Downloaded by [University of California, San Diego] at 16:33 22 March 2016
Gastrin Receptor Assay
label to rat gastric fundic mucosa. They used tritiated gastrin with a low specific radioactivity (60 Ci/mmol). Binding sites were described both on plasma membranes and on isolated cells, but the affinity (&; 9 nM) was not in the physiologic range. Three years later, Takeuchi et al. (11) described their gastrin receptor assay, using a crude plasma membrane fraction from rat fundic mucosa and iodinated gastrin with a high specific radioactivity as a tracer. High-affinity binding sites (Kd; 0 4 nM) were detected, and the gastrin receptor in the oxyntic mucosa was biochemically characterized (12). In 1984 Sol1 et al. (13) described specific high-affinity binding of '251-15leucine gastrin to dispersed canine fundic mucosal cells (13). Using elutriation to separate these cells by size, they were able to show that the binding correlated with the parietal cell content. Several laboratories have since reported modifications of the gastrin receptor binding assay. Different ligands and preparations of fundic mucosa have been used (Table I). Highaffinity binding sites have been demonstrated in the oxyntic mucosa in different species, including the rat (14), dog (13), rabbit (15), pig (16), and guinea-pig (17). Similar binding sites have been found in colon cancers (14,18), in some colon (14, 19) and stomach (20,21) cancers, but only inconstantly in duodenum (18). However, functional studies indicate that in the rat (22) and even in other species (23,24), the acid-stimulatory effect of gastrin may be
mediated by liberated histamine. In the rat histamine is produced (25) and released from the enterochromaffin-like (ECL) cell. In other species, too, the E C L cell produces histamine (26), and may be the source of the histamine participating in the stimulation of the acid secretion (23,24). It should be also recalled that gastrin has a specific trophic effect on the ECL cell in most species (27-30). Therefore, there must be a gastrin receptor on the E C L cell, and functionally there is no need for a gastrin receptor on the parietal cell (24). If the gastrin receptor is located only on the E C L cell (24), representing only a few per cent of the cells in the oxyntic mucosa (31,32), it is easily imagined that the receptor concentration may be too low to enable detection in binding experiments. In fact, this could be the explanation of the extraordinary difficulties in establishing a reproducible and accepted gastrin receptor assay. The principle of the gastrin receptor binding assay is the use of a biologically active labelled gastrin, which is incubated in vitro with a tissue receptor preparation. After the incubation period the hormone bound to the tissue is separated from the unreacted label by either centrifugation or filtration, and the bound radioactivity is counted. The development of a gastrin receptor binding assay, accordingly, involves several critical steps to be evaluated: the production of a stable gastrin tracer with preserved biologic activity, the preparation of a suitable tissue
Table 1. Biochemical characteristics of the gastrin receptor Ligand '251-G1-17 1ZSI-'51eu-G1-17t IZsI-'Me-G1- 17$ 1ZsI-'51eu-G2-17 '251-'51e~-G217
* Kd = dissociation constant. t Leu = leucine.
t
Nle = norleucine.
63
Fundic tissue preparation Plasma membrane fraction Isolated cells Isolated cells Isolated glands Detergent solubilized gastric mucosa
Kd* 0.4-1.1 0.46 0.07 2.3 300
Species reference Rat (14, 55)
Dog (13) Rabbit (15) Guinea-pig (17) Pig (16)
64
P. M . Kleueland & H . L. Waldum
preparation from the oxyntic mucosa, the conditions during incubation, and the correlation of binding to the biologic response of gastrin.
Downloaded by [University of California, San Diego] at 16:33 22 March 2016
THE LABELLED GASTRIN Gastrin circulates in plasma at a concentration in the lower pM range. The high affinity between gastrin and its receptor makes it necessary to produce labelled gastrin with high specific activity for use in binding studies. The radioactivity of labelled gastrin with a single '''1 molecule would be greater than for single substitutions with either "1 or "C by a factor of 72 and 32,000, respectively (4). Iz5I is accordingly the most suitable radionucleotide for producing labelled gastrin for receptor-binding studies. Highly enriched isotope preparations with a nearly 100% abundance of '?I (2150 Ci/mmol) are now available, and it is thus possible to produce monoiodinated tracers with about the same specific radioactivity (13. 15,33). The concentration of the tracer is determined by radioimmunoassay after identical immunoreactivity of the tracer is confirmed by comparison with unlabelled gastrin (that is, they have superimposable binding curves). The radioactive gastrin, however, may lose biologic activity as a result of the labelling process (34) or by the mere presence of the I atom itself (35). Data concerning biologic activity of the radioiodinated gastrins used for receptor binding are therefore of great importance. The assessment of biologic activity of labelled gastrin, however, has been cumbersome and difficult. The usual in vivo bioassays for gastrin have several disadvantages: they are laborious, lack precision, may be influenced by metabolism and excresion, and require large amounts of label (36). Therefore, macroscale iodinations (mixtures of '"I and '?'I) producing gastrin of low specific radioactivity have been performed to test biologic activity of the iodinated gastrin (37). The tracer itself, with high specific activity exposed to radioactivity during iodination and storage and used for receptor binding studies, was not tested (11, 14). The bioassay of gastrin. however, was recently significantly improved by the development of the totally isolated vascularly
perfused rat stomach (38,39). This bioassay is sensitive to gastrin in physiologic concentrations, requires only small amounts of gastrin, and was found useful for direct testing of the routine laboratory tracers with high specific activity (33). Quite recently, this model has been further improved by determination of the immediate histamine release into the venous drainage (40). Chloramine-T oxidation has been the method most widely used for iodination of gastrin (41). However, it may destroy the molecule, leading to loss of biologic activity (34). Iodination performed even with short reaction times and at low chloramine-T/gastrin molar ratios may give biologically inactive gastrin tracers (Kleveland and Waldum, unpublished observations) (Fig. 1). Chloramine-T oxidizes methionine and probably also the tryptophan residue, resulting in a total loss of biologic activity (34, 42,43). This is somewhat at odds with earlier studies, which reported that ultrashort chloramine-T oxidation does preserve the biologic activity of the gastrin tracer (37). Solid-phase iodination with Iodogen may give less damage to the gastrin molecule and thus retain biologic activity (44). Tracers prepared with Iodogen have been demonstrated to retain biologic gastrin activity in gastric fistula rats (36), dogs (14), and in the totally isolated vascularly perfused rat stomach (33) (Fig. 1). Nevertheless, Iodogen may also oxidize methionine, but probably only to its sulphoxide, which is biologically active (43). This could explain the minor difference in the biologic activity of iodinated gastrin prepared by chloramine-T and that prepared by Iodogen iodinations. To prevent the oxidation of methionine, analogues using leucine o r norleucine substitutions for 15-methionine have been synthesized (45,46). These gastrin analogues are reported to have full biologic activity and to be more resistant to oxidation (11,13,37). They seem to be an attractive alternative when preparing gastrin tracers for receptor binding studies. Moreover, 15-norleucine gastrin is chemically the closest to S m e t h i o n i n e gastrin and therefore theoretically the most ideal analogue (43). Whereas 1251-gastriniodinated by a chloramineT method tends to be rather unstable and give
Gastrin Receptor Assay
G
IMX
1-17
I I I I I
T
I
A
I I
Downloaded by [University of California, San Diego] at 16:33 22 March 2016
65
T
----.-p-.-....p. .+....... .....I.
I
Y
"2 0
40
I
60
.....I.
1
, a l p ]
80
100
TIME ( m i d Fig. 1. A comparison between the biological effect of G1-17 (0----0). "'I-G 117 (Iodogen) ( 0 - 0 1 , and '251-G1-17(chloramine-T) (W-W) at a concentration of 65 pM on the acid secretion by the totally isolated vascularly perfused rat stomach co-stimulated with isobutylmethylxanthine at a concentration of 50 pM. For comparison the base-line ( A---- A ) and maximal gastrin stimulated acid secretion (0----U) are given (Ref. 33). Chloramine-T iodination was performed as described in Ref. 37.
low specific binding (13), 1251-gastrinprepared by Iodogen is reported to give reproducible high specificbinding (14). Iodinated 15-leucine (11, 13) and 15-norleucine analogues (15) also give biologically active gastrin tracers suitable for receptor studies. Similar Kd and number of binding sites on isolated canine parietal cells have been reported with iodinated gastrin and gastrin analogues prepared by the Iodogen method (43). RECEPTOR PREPARATIONS Gastrin receptors have mainly been studied in a plasma membrane fraction from homogenized oxyntic mucosa or on dispersed cells and, recently, o n gastric glands from the oxyntic mucosa. PLASMA MEMBRANE FRACTIONS Plasma membrane fractions are easy to prepare
and can be stored and used for screening
for receptors in most organs. By different centrifugation techniques combined with enzyme markers, plasma membranes of various degrees of purification from oxyntic mucosal homogenates have been prepared (9,11,14). However, there have been some major problems. The homogenization and purification steps are often time-consuming and rather crude mechanical procedures that expose the receptors to liberated proteolytic enzymes. Retained proteolytic activity in the final receptor preparation can affect both the receptor and the labelled gastrin during incubation (10). The main problem, however, seems to be the heterogeneity of the cells in the oxyntic mucosa. Plasma membranes from dispersed fundic mucosal cells enriched with a particular cell type have not, to our knowledge, been successfully used. Since the cells possessing the gastrin receptor may only constitute a small
Downloaded by [University of California, San Diego] at 16:33 22 March 2016
66
P. M. Kleuelund & H . L. Wuldum
fraction of the total fundic mucosal tissue, the non-specific binding will tend to be too high to enable detection of the specific billding. However, in 1979 a crude plasma membrane fraction from fundic mucosa was reported suitable for gastrin receptor binding by Takeuchi et al. ( 1 I ) . The gastrin receptor was extensively studied and biochemically characterized (12). The method has, however, never been reproduced by other laboratories. Several significantly different modifications of the assay have been reported to detect specific binding (14, 18). The poor reproducibility and inconsistency of the results make the method rather uncertain. DISPERSED CELLS A N D GLANDS Isolated fundic mucosal cells are easy to prepare and offer the advantage that the cell mixture can be fractionated in accordance with the cell type of interest (13). It is not possible to assess acid secretion directly from isolated parietal cells, but it may be done indirectly by the incorporation of the weak base aminopyrine (47). Unfortunately, gastrin has been found to be a weak and inconstant stimulator of the aminopyrine uptake in isolated parietal cells (48) and isolated gastric glands (47). Moreover. the biologic activity of gastrin on the cellular level has been difficult to assess. since its second messenger has not yet been established (49). The treatment of the tissue with pronase and/or collagenase may alter the biologic function of the receptors (4). Pronase has a more general proteolytic activity than collagenase and may be expected to damage the receptor more easily. To isolate cells from a thick-walled stomach like the rat stomach. i t is necessary to use pronase (SO). Nevertheless, parietal cells prepared by the use o f either collagenase and/or pronase in addition to ethylenediaminetetraacetic acid (EDTA) to bind Ca*+ have retained responsiveness towards histamine and carbacholine (51). Gastrin. however. has no or only faint stimulatory effect o n the aminopyrine uptake in isolated parietal cells and gastric glands (47, 48. 51). Sol1 et al. (13). using isolated canine parietal cells enriched by elutriation. have confirmed some of the binding data obtained in the rat plasma membrane
fraction. They used chloramine-T-labelled 1 Sleucine gastrin and correlated the specific binding with the accumulation of aminopyrine in parietal cells. Binding of gastrin, however. was also detected in fractions containing smaller cells, and the parietal cell fraction used was not completely pure (13). Isolated gastric oxyntic glands have recently been reported to possess high-affinity binding sites for gastrin (17), but this method does not establish the cellular localization of the receptor. Gastrin-binding proteins have quite recently been identified in detergent extracts from whole gastric fundic mucosa (16), isolated parietal cells (52). and gastric glands (17) by covalent crosslinking of iodinated G2.17 to elucidate the molecular structure of the gastrin receptor. The gastrin proteins, however, had very low affinities for the gastrin probe (16). INCUBATION The specific binding has been estimated as the difference between binding without (total binding) and with a thousandfold excess of unlabelled gastrin (non-specific binding). A5 for other peptide hormones incubation damage is a major factor to be avoided when establishing stable binding to a tissue preparation (53). Incubation damage can potentially affect both label and receptor. It is usually minimal when using whole intact cells and greatest with plasma membranes from whole homogenates (53). Accordingly, Soil et al. (13) reported isolated canine cells to degrade only 11% of their chloramine-T-iodinated '51eucine-G,.,,, and Ramani et al. (17) reported a 25% degradation of an identical but less purified label with isolated guinea-pig gastric glands after 30 min at 37°C. In the latter study the activity was assumed to be proteolytic, but protease inhibitors were reported to be without any effect (17). Substantial incubation damage has recently been demonstrated in the standard crude 270- to 30,000-g crude plasma membrane fraction from fundic mucosal homogenates (18). The original preincubation and incubation conditions at 30°C. introduced by Brown & Gallagher (54) and later
Downloaded by [University of California, San Diego] at 16:33 22 March 2016
Gastrin Receptor Assay
adopted by Takeuchi et al. (11) were later shown to give variable and mainly non-specific binding (36). Althoughthe degradation was demonstrated to be partially proteolytic, protease inhibitors were ineffective (18). The damage to the label and the binding site was only controlled by reducing the incubation temperature to 15"Cand omitting the preincubation. Thus, reproducible specific binding t o fundic plasma membranes with a Kd of 0.8 nM (Scatchard analysis) was detected (18). This variant of the assay was later reproduced by Rossowski et al. (55). Singh et al. (14), also by omitting the preincubation period and by repetitive washing of the fundic mucosal homogenate, have recently reported high-affinity binding to fundic mucosa at 30°C, using Iz5I-gastrin iodinated with the help of Iodogen. Controls for incubation damage, however, were not presented. Damage to IZ5Igastrin with plasma membranes from fundic homogenates at 30°C was noticed already in the original assay presented by Brown & Gallagher (54). Controls for potential damage were not included in the studies of Takeuchi et al. ( l l ) , who adopted this method for characterization of the gastrin receptor in rat fundic mucosa (12). Damage to the label and loss of receptor activity may seriously affect calculations of binding affinities (&) and the number of binding sites. CORRELATION BETWEEN BINDING A N D BIOLOGIC EFFECT The direct measurement of acid is only possible in intact fundic mucosa. Any correlation between the binding of labelled gastrin to plasma membranes and a biologic effect it has so far not been possible to evaluate. Specific binding to rat fundic plasma membranes was recently indirectly shown to correlate with biologic activities of the label in the totally isolated vascularly perfused rat stomach during storage of an Iodogen-labelled '"I-gastrin (56). This study indicates that the specific binding does reflect an interaction with the receptor (56). Other criteria for true receptor binding, however, were not satisfactorily fulfilled, making the interpretation difficult. The strongest evidence for a direct correlation
67
between specific binding and the biologic effect of gastrin has been reported on isolated canine fundic mucosal cells (13). Specific binding of a '251-labelled 15-leucine gastrin analogue prepared with chloramine-T was shown to be correlated with the accumulation of aminopyrine when the '*'I label of the gastrin analogue was used as a stimulant (13). Although some specific binding was observed in a fraction with small cells, the binding was mainly confined to fractions containing the parietal cells (13). The authors and many others have considered this study to be major proof of the existence of a functional gastrin receptor on the parietal cell, at least in the dog. The data, however, are still discussed in view of the inconsistent and weak effect of gastrin and the lack of differential counting and characterization of the cells present in the parietal cell fraction used. The stimulatory effect could therefore possibly be accounted for by coelution of parietal and histamine-producing cells. Histamine-producing cells have recently been reported to exist in the dog fundic mucosa as well (57). Nevertheless, quite recently, monoclonal antibodies have been produced by immunization with canine parietal cell fractions and postulated to be directed towards a gastrin receptor on the parietal cell (58). The monoclonal antibodies inhibited the binding of labelled gastrin and reduced aminopyrine uptake in the isolated canine parietal cells used (58). CONCLUSION The gastrin receptor binding assay apparently still does not fulfil the criteria for true receptor binding. Both the cell type possessing the receptor and its second messenger are still under discussion. A new non-peptidic gastrin antagonist (59) will probably be a useful tool to characterize and localize the gastrin receptor further.
REFERENCES 1. Walsh J H , Grossmann MI. Medical progress:
gastrin. N Engl J Med 1975, 292, 13241334 2. Gregory RA, Tracy HJ. The preparation and properties of gastrin. J Physiol (Lond) 1961, 156, 523-543
Downloaded by [University of California, San Diego] at 16:33 22 March 2016
68
P. M . Kleveland & H. L. Waldum
3. Gregory RA, Hardy PM, Kenner GW, Sheppard RC. Structure of gastrin. Nature 1964, 204, 931933 4. Gardner JD. Receptors for gastrointestinal hormones. Gastroenterology 1979, 76, 202-214 3 . Catt KJ, Dufau ML. Introduction: The clinical significance of peptide hormone receptors. Clin Endocrinol Metabol 1983, 12, XI-XLIV 6.Scatchard G . The attractions of proteins for small molecules and ions. Ann NY Acad Sci 1949. 51, 66&672 7. Akara T, Cheng V-JK. A simple method for the determination of affinity and binding site concentration in receptor binding studies. Biochim Biophys Acta 1977. 470, 412-423 8. Lin S-Y, Goodfriend TL. Angiotensin receptors. Am J Physiol 1970. 218. 1319-1328 9. Lewin M. Soumarmon A, Bali JP, et al. Interaction of %labelled synthetic human gastrin I with rat gastric plasma membranes. Evidence for the existence of biologically reactive gastrin receptor sites. FEBS Letters 1976, 66. 168-172 10. Soumarmon A. Cheret AM, Lewin MJM. Localization of gastrin receptors in intact isolated separated rat fundic cells. Gastroenterology 1977. 73. 90tL903 1 1 . Takeuchi K. Speir GR, Johnson LR. Mucosal gastrin receptor. I. Assay standardization and fulfillment of receptor criteria. Am J Physiol 1979. 237. E284E29.1 12. Johnson LR. Gastrin receptor assay. Meth Enzymol 1985. 109, 56-64 13. Sol1 AH, Amirian DA, Thomas LP. Reedy TJ, Elashoff JD. Gastrin receptors on isolated canine parietal cells. J Clin Invest 1984, 73. 1434-1447 1.2. Singh P, Rae-Venter B, Townsend CM Jr. Khalil T. Thompson JC. Gastrin receptors in normal and malignant gastrointestinal mucosa: age-associated changes. Am J Physiol 1985. 249, G761-769 IS. Magous R. Bali J-P. High affinity binding sites for gastrin on isolated rabhit gastric mucosal cells. Eur j Pharmacol 1982. 82. 17-54 16. Baldwin GS. Chandler R, Scanlon DB, Weinstock J . Identification of a gastrin binding protein in porcine gastric mucosal membranes by covalent cross-linking with iodinated gastrin 2-17. J Biol Chem 1986. 261. 12252-12257 17 Kainani N, Praissman M. Molecular identification and characterization of the gastrin receptor in guinea pig gastric glands. Endocrinology 1989. 124. 1881-1887 I X Klrveland PM. Waldum HL. Binding and degradation of "'I-gastrin by plasma membranes from homogenized rat gastric mucosa. %and J Gastroenterol 1986. 21. 547-555 19 Upp JR Jr. Singh P, Townsend CM, Thompson JC. Clinical significance of gastrin receptors in human gastric colon cancers. Cancer Res 1989. 49. 48% 492 20 Kumamoto T. Gastrin receptors in human gastric scirrhous carcinoma. Gastroenterol Jpn 1988. 23. 384-389 21 Wein\tock J. Balwin GS Binding of gastrin,, to
human gastric carcinoma cell lines. Cancer Res 1989, 48, 932-937 22. Sandvik AK, Waldum HL, Kleveland PM, Schulze S ~ g n e nB. Gastrin produces an immediate and dose-dependent histamine release preceding acid secretion in the rat. Scand J Gastroenterol 1987, 22, 803-808 23. Waldum HL, Sandvik AK, Brenna E , Petersen H. The gastrin-histamine sequence in the regulation of gastric acid secretion. Gut 1990 (in press) 24. Waldum HL, Sandvik AK. Histamine and the stomach. Scand J Gastroenterol 1989, 24, 13&139 25. Hikanson R, Owman C. Argyrophilic reaction of histamine-containing epithelial cells in murine gastric mucosa. Experientia ,1969, 25, 626 26. HikansonR,BottcherG, EkbladE.etal. Histamine in endocrine cells in the stomach. Histochemistry 1986, 86, 5-17 27. Larsson H, Carlsson E, Mattsson. et al. Plasma gastrin and gastric enterochromaffin-like cell activation and proliferation. Gastroenterology 1986. 90, 391-399 28. Tielemans Y , Axelson J. Sundler F, Willems G, Hikanson R . Serum gastrin concentration affects the self replication rate of the enterochromaffinlike cells in the rat stomach. Gut 1990. 31, 274-278 29. Borch K, Renvall H, Liedberg G . Gastric endocrine cell hyperplasia and carcinoid tumors in pernicious anemia. Gastroenterology 1985, 88. 638-648 30. Bordi C, Coconi G , Togni R. Vezzadini P, Missale G. Gastric endocrine cell proliferation. Association with Zollinger-Ellison syndrome. Arch Patholl974, 98. 274278 31. Hikanson R , Larsson LI, Liedberg G. Oscarson J , Sundler F, Vang J. Effects of antrectomy or portacaval shunting on the histamine-storing endocrinelike cells in oxyntic mucosa of rat stomach. A fluorescence histochemical, electron microscopic and chemical study. J Physiol 1976, 259, 785-800 32. Rubin W, Schwartz B. Electron microscopic radiographic identification of the ECL cell as the histamine-synthesizing endocrine cell in the rat stomach. Gastroenterology 1979. 77, 458-467 33. Kleveland PM, Waldum HL, Bjerve KS. F j ~ s n e HE. Bioassay of gastrin using the totally isolated vascularly perfused rat stomach. A biomodel sensitive to gastrin in physiological concentrations. Scand J Gastroenterol 1987. 21, 945-950 34. Stagg BH, Temperley JM, Rochman H, Morley JS. Biological activity of iodinated gastrin. Nature 1970. 228, 58-59. 35. Kahn RC. Membrane receptors for polypeptide hormones. Meth Membr Biol 1975, 3. 81-146 36. Kleveland PM. Haugen SE, Waldum HL. The preparation of bioactive '"I-gastrin, using lodogen as oxidizing agent, and the use of this tracer in receptor studies. Scand J Gastroenterol 1985. 20, 569-576 37. Dockray GH. Walsh JH, Grossman MI. Biological activity of iodinated gastrins. Biochem Biophys Res Commun 1976. 69. 339-345 38. Kleveland PM. Haugen SE. Sandvik AK. Waldrum HL The effect ofu pentagastrin on the gastric
Downloaded by [University of California, San Diego] at 16:33 22 March 2016
Gastrin Receptor Assay secretion by the totally isolated vascularly perfused rat stomach. Scand J Gastroenterol 1986, 21, 379384 39. Kleveland PM, Haugen SE, Waldum HL. Effect of pentagastrin on the gastric acid secretion in the totally isolated vascularly perfused rat stomach stimulated with the phosphodiesterase inhibitor isobutyl methylxanthine. Scand J Gastroenterol 1986, 21, 577-584 40. Sandvik AK, Waldum HL. Rat gastric histamine release: a sensitive gastrin bioassay. Life Sci 1990, 46, 453-459 41. Stadil F, Rehfeld JF. Preparation of '251-labelled synthetic gastrin human gastrin I for radioimmunoanalysis. Scand J Clin Lab Invest 1972, 30, 361-368 42. Mourier G , Moroder L, Previero A. Prevention of tryptophan oxidation under iodination of tyrosyl residues in peptides. Z Naturforsch 1984,39b, 101104 43. Seet L, Fabri L, Nice EC, Baldwin GS. Comparison of iodinated (NleIs)- and (Met ")-gastrin,, prepared by reversed-phase HPLC. Biomed Chromat 1987, 2, 159-163 44. Fraker PJ, Speck JC Jr. Protein and cell membrane iodination with a sparingly soluble chloromide 1,3,4,6-tetrachloro-3a,6a-diphenylglycoluril.Biochem Biophys Res Commun 1978, 80, 849-857 45. Wunsch E , Deimer KH. Zur Synthese von HumanLittle-Gastrin-I und dessen Leucin-15 analogue. Hoppe-Seylers Z Physiol Chem 1972, 353, 12551258 46. Moroder L, Gohring W, Nyfeler R, Scharf R, Thamm P, Wendelberger G. Zur Synthese von Human-Little-Gastrin-I und dessen Leucin-15-, Norleucin- und Methionin-15-Analoga. HoppeSeylers Z Physiol Chem 1983, 3364, 157-171 47. Berglindh T , Helander HF, Obrink KJ. Effect of secretagogues on oxygen consumption, aminopyrine accumulation and morphology in isolated gastric glands. Acta Physiol Scand 1976, 97, 401-414 48. Sol1 AH. Secretagogue stimulation of "C-aminopyrine accumulation by isolated canine parital cells. Am J Physiol 1980, 238, G366G375
69
49. Morriset J. Gastrointestinal hormone receptors in the gut: localization, characterization, modulation, and ontogeny. In: Lebenthal E , ed. Human gastrointestinal development. Raven Press, New York, 1989, 99-122 50. Lewin M, Cheret AM, Soumarmon A , Girodet J. Methode pour I'isolement et la tri cellules de la muoqueuse fundique de rat. Biol Gastroenterol (Paris) 1974, 7, 139-144 51. Dial E, Thompson WJ, Rosenfeld GC. Isolated parietal cells: histamine response and pharmacology. J Pharmacol Exp Ther 1981, 219-585-590 52. Matsumoto M, Park J, Yamada T. Gastrin receptor characterization; affinity crosslinking of the gastrin receptor on canine gastric parietal cells. Am J Physiol 1987, 252, G142-G147 53. Ryan RJ, Lee CY. The role of membrane bound receptors. Biol Reprod 1976, 14, 1 6 2 9 54. Brown J, Gallagher ND. A specific gastrin receptor site in the rat stomach. Biochem Biophys Acta 1978, 538,42-49 55. Rossowski WJ, Ozden A, Ertan A, Maumus M, Arimura A. Somatostatin, gastrin, and cholinergic muscarinic binding sites in rat gastric, duodenal, and jejunal mucosa. Scand J Gastroenterol 1988, 23, 717-725 56. Kleveland PM, Waldum HL. The storage time of monoiodinated gastrin affects the biological activity and binding to rat fundic plasma membranes similarly, whereas the immunoreactivity is less affected. Scand J Gastroenterol 1987, 22, 390-396 57. Chuang CN, Toomey M, Chen MC, Sol1 AH. Histamine stores in the canine oxyntic mucosa: differentiation of two cell populations. Gastroenterology 1990, 98, A31 58. Mu F-T, Baldwin G , Weinstock J , Stockman D, Toh BH. Monoclonal antibody to the gastrin receptor on parietal cells. Proc Natl Acad Sci USA 1987, 84, 2698-2702 59. Huang SC, Zhang L, Chiang H-C, et al. Benzodiazepine analogues L365.260 and 364,718 as gastrin and pancreatic CCK receptor antagonists. Am J Physiol 1989, 257, G169-G174
