Immunohistochemical Differentiation of Gastrin and Cholecystokinin in Gastrointestinal Tract of Chickens

Immunohistochemical Differentiation of Gastrin and Cholecystokinin in Gastrointestinal Tract of Chickens V. MARTINEZ, A. RODRIGUEZ-MEMBRILLA, M. JIMENEZ, E. GONALONS, and P. VERGARA1 Department of Cell Biology and Physiology, Veterinary Faculty, Universidad Aut6noma de Barcelona, Bellaterra, 08193 Barcelona, Spain

1993 Poultry Science 72:232S-2336

INTRODUCTION

Cholecystokinin (CCK) is a peptide that is widely distributed both in the nervous system and in the digestive tract of mammals (Rehfeld, 1989). Cholecystokinin and gastrin are very similar structurally in their active moiety, and both have the Cterminal pentapeptide in common. Therefore, both peptides are included as members of the same family with a common evolutionary origin (Larsson and Rehfeld, 1977; Dockray, 1988). In mammals, both

Received for publication February 12, 1993. Accepted for publication July 15, 1993. J To whom correspondence should be addressed.

peptides and their forms have been characterized in several species, as has their distribution (Dockray and Gregory, 1989; Rehfeld, 1989). Gastrin is present in endocrine cells of the antral mucosa (>90%) and proximal d u o d e n u m . Cholecystokinin has a more heterogeneous presence: in the central nervous system, gastrointestinal tract, in both endocrine cells and nervous fibers, and in other organs like the urinary bladder and lung. In the gastrointestinal tract, CCK is present in endocrine cells in the duodenum, jejunum, and ileum, with the highest density in the jejunum. The presence of CCK is scarce in nervous fibers located mainly in the rectum, where it is collo-

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ABSTRACT The presence of cholecystokinin (CCK) in the gastrointestinal tract of chickens has not been well demonstrated, although immunological and chromatographic techniques have shown the presence of intestinal gastrinCCK-like factors. Recently, a new peptide, structurally related to mammalian CCK, but with a gastrin-like activity, has been isolated from the digestive tract of chickens. The objective of this work has been: 1) to study the presence of gastrin-CCK-like immunoreactivity (IMR) in the digestive tract of chickens; 2) to distinguish chicken gastrin from CCK; and 3) to establish their distribution using specific antibodies. Tissue specimens from the proventriculus, gizzard, pylorus, duodenum, jejunum, ileum, ceca, and rectum were studied using indirect immunofluorescence procedures. The antibodies used were: 1) an antibody specific against the C-terminal pentapeptide common to gastrin and CCK; 2) one specific against CCK-33; and 3) one specific against chicken gastrin. Their use allowed the differentiation of two cellular populations which showed different affinities for the antibodies, indicating the presence of a gastrin-like peptide in the antrum and another CCK-like peptide in the small intestine, with the highest concentration in the proximal ileum. Immunoreactivity was not found in any other studied area. Two different peptides of the gastrin-CCK family are present in the chicken's gastrointestinal tract. However their differentiation and identification are more difficult than in mammals due to the greater structural similarities of these peptides in birds. (Key words: chicken gastrin, cholecystokinin, immunohistochemistry, stomach, intestine)

GASTRIN AND CHOLECYSTOKININ IN CHICKEN GUT

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injection of sodium pentothal and specimens from proventriculus, gizzard, pylorus, proximal and distal duodenum, proximal jejunum, distal jejunum (10 cm orad to vitelline diverticulum), proximal ileum (10 cm aborad to vitelline diverticulum), distal ileum (5 cm from ileo-ceco-colic junction), cecum, and rectum were excised. The tissues were immediately fixed in a solution of .4% (wt/vol) recrystallized P-benzoquinone in PBS (NaCl, 8 g/L; KC1, .2 g/L; Na2HP04, 1.15 g/L; KH2P04 pH 7.4) for 4 h, transferred to PBS with 20% sucrose (wt/vol) for 24 h, then frozen in liquid nitrogen and kept at -80 C for further study. Cryostat sections were cut at 10 /an thickness and stained by the indirect immunofluorescence method of Coons (1956). Antisera used and their dilutions are summarized in Table 1. The sections were incubated for 16 to 24 h at 4 C with the first antiserum diluted in PBS containing .1% BSA and .01% sodium azide. The secondary antiserum was goat anti-rabbit IgG conjugated to fluorescein,2 used at a dilution of 1:16 in PBS and incubated with tissue sections in a moist chamber at room temperature for 30 min. After PBS wash, sections were mounted in PBS-glycerol (1: 9) and examined using a fluorescence microscope.

MATERIALS AND METHODS

In order to determine the specificity of the primary antisera, each antiserum was absorbed with either CCK-8, chicken gastrin-36 nonsulfated form, or porcine CCK-33 in a final concentration of 20 /xg/ mL. Then the absorbed and unabsorbed antisera were tested for stainability using

Six- to 8-wk-old chickens of either sex (n = 6) were killed with an intravenous

2

Sigma Chemical Co., St. Louis, MO 63178-9916.

Antisera Characteristics of antisera and their dilutions are shown in Table 1. Antiserum L48 recognizes the C-terminal common to gastrin and CCK. Antiserum L293 was raised against chicken gastrin-10 (Fragments 27 to 36 of chicken gastrin). Antiserum A-CCK is specific for CCK, in that it does not recognize the C-terminal common to gastrin and CCK. At least four sections of each block were incubated for each antiserum. Absorption Experiments

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cated with other neuropeptides (Furness et al, 1984). In avian species there are a smaller number of studies, and less is known regarding the gastrin-CCK family. A CCKlike immunoreactivity has been described in pigeon brain (Vanderhaeghen et al, 1975), chicken and duck duodenum (Larsson and Rehfeld, 1977), and turkey brain and duodenum (Dockray, 1979). Recently, a 36-amino acid residue has been isolated from the chicken antrum, a so-called small area about .5 cm long at the junction of the gizzard and duodenum (Dimaline et al, 1986). This molecule is similar to the mammalian CCK but shows an activity similar to gastrin, both in mammals and birds (Vigna, 1984; Dimaline and Lee, 1990). In the same way, CCK-8 has recently been isolated from the chicken brain, and its sequence is identical to mammalian CCK (Fan et al, 1987). Intravenous infusion of CCK inhibited gastric motility with an increase of duodenal activity (Savory et al, 1981; Martinez et al, 1993), suggesting a physiological role for CCK in birds. There are also a few studies of gastrinCCK immunoreactivity in the avian digestive tract. Most of these studies were done with nonspecific antibodies, and therefore differentiation between gastrin and CCK in the digestive tract was not possible (Polak et al, 1974; Yamada et al, 1979; Rawdon and Andrew, 1981). There is no study of the distribution of chicken gastrin in the gastrointestinal tract of chickens. The aim of this study was therefore: 1) to study the distribution of chicken gastrin in the gastrointestinal tract by means of a specific antibody; 2) to establish the presence of CCK and its distribution with a special emphasis on its differentiation from chicken gastrin; and 3) to quantify the presence of these gastrin and CCK-like peptides in the gastrointestinal tract of chickens.

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MARTINEZ ET AL. TABLE 1. Antisera used

Antibody Source L48 L293 A-CCK

3

G. J. Dockray G. J. Dockray3 Peptide Institute4

Raised against1-2

Specificity

CCK-8 II cG-10 I porcine CCK

C-terminal common to gastrin and CCK 1:200 C-terminal of chicken gastrin 1:50 CCK-33 1:200

Dilution

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CCK = cholecystokinin; cG = chicken gastrin. I, II = nonsulfated and sulfated form, respectively. SG. J. Dockray, Physiology Department, University of Liverpool, P.O. Box 147, Liverpool L69 3BX, UK. 4 Peptide Institute, 4-1-2 Ina Minoh-Shi, Osaka 562, Japan. 2

Cell Counting

However, specific differences were shown when the antibodies were absorbed with CCK octapeptide, chicken gastrin-36, or CCK-33 (Table 2).

Nucleated and specifically stained cells Antrum were counted in at least 10 fields per section No immunoreactive cells were observed using a 12.5x eyepiece and a 25x objective when sections were incubated with L48, the lens. Results are expressed as x i SEM. antibody against the C-terminal pentapeptide of the gastrin-CCK. A very high density Controls of immunoreactive cells (117 ± 40 cells per field, 325x magnification) were observed In order to validate the specificity of the when the sections were incubated with the immunoreactivity, the primary or secon- specific chicken gastrin antiserum (L293) dary antisera were omitted or replaced with (Figure 3A). Cells were allocated throughnormal rabbit serum. out the whole thickness of the antral mucosa (Figure 1A). When antral sections were incubated with the A-CCK antiserum, a less intense immunoreactivity was obRESULTS served in the same cells that were stained Cells containing immunoreactivity to with the L293 antiserum, indicating that the gastrin-CCK peptides were stained with a CCK-33 antibody also recognized some bright fluorescence, which was restricted areas of the gastrin-like peptide present in to the cytoplasm. Stained cells were often the antrum. Chicken gastrin-36 completely pyramidal with an apical pole that ap- blocked the immunoreactivity to L293 and peared to project into the glandular lu- to A-CCK (Figure 3B). Cholecystokinin-33 men. They were confined to the mucosa of completely blocked immunoreactivity to Athe antrum, duodenum, and small intes- CCK and significantly reduced that to L293 tine (Figure 1), and they were not ob- (Figure 3C). The o c t a p e p t i d e of served in the proventriculus, gizzard, cholecystokinin (CCK-8) prevented L293 ceca, or rectum. In the small intestine, the reaction and reduced significantly that of highest concentration was observed A-CCK (Figure 3D; Table 2). around the vitelline diverticulum, decreasing progressively both proximally and distally. A relative distribution of im- Duodenum munoreactive cells is represented in FigScarce cells reacting to L48, L293, and Aure 2. Only very rarely were scarce CCK antisera were observed both in the immunoreactive nervous fibers observed. proximal and distal duodenum (Table 2). Both L293 and A-CCK antisera reacted They were located mainly in the villi with the same cells in all the studied areas. (Figure 4A). Chicken gastrin-36 did not

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consecutive intestinal sections from each chicken.

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GASTRIN AND CHOLECYSTOKININ IN CHICKEN GUT

Small Intestine Cells highly reacting to L48, L293, and ACCK were observed in the villi along the small intestine (Table 2; Figure 5A). Immunoreactivity was prevented by the previous absorption of the antisera with

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prevent staining when the sections were incubated with the A-CCK antisera, suggesting that some cells contained a peptide different from chicken gastrin (Figure 4B). Cholecystokinin-33 completely abolished immunoreactivity to the antisera (Figure 4C). In addition CCK-8 did not prevent immunostaining of L293 or A-CCK (Figure 4D). In those preparations in which it was possible to visualize the transition area between the antrum and the duodenum it was clear that, whereas chicken gastrin prevented the reaction of the antibodies against the peptide in the antrum, duodenal cells remained immunoreactive.

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MARTINEZ ET AL. Jejunum

Ileum

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Duodenum FIGURE 2. Diagrammatic illustration of the alimentary tract of the chicken with distribution of cells staining for gastrin100.

CCK-33 (Figure 5B). Chicken gastrin-36 (Figure 5C) or CCK-8 (Figure 5D) did not prevent the immunoreaction to A-CCK antiserum. The highest concentration of cells was found in the proximal ileum (3.37 ± 1.21 cells per field, 325x magnification) and in the distal jejunum (2.55 ± .91 cells per field). Scarce cells were observed in proximal jejunum and distal ileum (Figure 2). DISCUSSION

This study demonstrated immunohistochemically the presence of two different peptides of the family gastrin-CCK in the gastrointestinal tract of chickens. The antrum is very rich in endocrine cells containing chicken gastrin, whereas in the small intestine there is a peptide more closely related to mammalian CCK. There have been several attempts to identify gastrin-CCK peptides in the avian gastrointestinal tract either by chromatographic techniques (Dockray, 1979), radioimmunoassay (Shulkes et al, 1983), or immunohistochemistry (Polak et al, 1974;

Yamada et al., 1979). However these studies used antisera against the Cterminal common to both gastrin and CCK. Under these conditions, cells containing peptides of the gastrin-CCK family were identified both in the stomach and the intestine. Rawdon and Andrew (1981), using antibodies against several CCK fragments, demonstrated that there were two different populations of cells that contained gastrin-CCK in the antrum and ileum of chicks. In the current study, the use of an antiserum for chicken gastrin, a peptide recently isolated from the chicken antrum (Dimaline et al, 1986), together with a specific antibody for the uncommon residue of the mammalian CCK plus a systematic absorption of the antibodies with several related peptides, has allowed a clear identification of two different populations of endocrine cells containing gastrin-CCK peptides. In the antrum, in agreement with other studies, the peptide seems to be chicken gastrin. However in the intestine, chicken gastrin was unable to prevent immunoreactivity to the A-

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Ileo-ceco-colic junction

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GASTRIN AND CHOLECYSTOKININ IN CHICKEN GUT

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FIGURE 4. Immunofluorescence micrographs of consecutive sections of chicken duodenum incubated with A-CCK. A) Control. B) Antiserum preabsorbed with chicken gastrin-36. C) Antiserum preabsorbed with porcine CCK-33. D) Antiserum preabsorbed with CCK-8. Scale: 20 /an.

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MARTINEZ ET Al.

strated by isolation studies, chicken gastrin in the antrum is larger than 10 amino acids. It is likely to be a 36-amino-acid residue as described by Dimaline et al. (1986). It also indicates that the amino terminal segment of the molecule has some similarities in amino acid sequence to Fraction 1-28 of porcine CCK. Antiserum L48 did not react to cells in the antrum whereas the same antibody reacted to antral cells of 1-day-old chicks (Rawdon and Andrew, 1981). In another series of studies (data not shown), antrum was incubated with another antiserum, also raised to CCK-8, and the same results were obtained. There is no plausible explanation for this difference from the results of Rawdon and Andrew (1981). Functional studies have demonstrated that chicken gastrin, in spite of being very close in amino acid sequence to CCK, stimulates gastric but not pancreatic secretions both in mammals and birds (Vigna, 1984; Dimaline and Lee, 1990). In studies on motility (Martinez et al, 1993), CCK and chicken gastrin have different effects.

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FIGURE 5. Immunofluorescence micrographs of consecutive sections of proximal ileum of chicken incubated with A-CCK. A) Control. B) Antiserum preabsorbed with porcine CCK-33. C) Antiserum preabsorbed with chicken gastrin-36. D) Antiserum preabsorbed with CCK-8. Scale: 20 (im.

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CCK antibody, indicating that the peptide present there is different from chicken gastrin and is likely to be a CCK-like peptide. In this sense the current results agree with those of Rawdon and Andrew (1981). The inability to identify the different peptides in spite of the use of specific antibodies against chicken gastrin and CCK is remarkable. However according to Larsson and Rehfeld (1977), CCK and gastrin in the avian gastrointestinal tract should be of greater similarity than those in mammals, and more closely related to mammalian CCK than to mammalian gastrin. Actually, CCK is phylogenetically older than gastrin and the amino acid sequence of chicken gastrin has more similarities to mammalian CCK than to mammalian gastrin. In addition, L293 was raised against chicken gastrin-10, and this sequence of amino acids has six amino acids in common with CCK-8, crossreacting in part with the C-terminal segment of CCK. The fact that A-CCK also reacted with antral cells indicates that, as demon-

GASTRIN AND CHOLECYSTOJONIN IN CHICKEN GUT

gastrin, respectively, are present in the gizzard are surprising. It is possible that those authors pooled together the gizzard and the antrum and considered them to be gizzard. The fact that the highest concentration of CCK is in the proximal ileum could be related to the functional role of CCK. In mammals, CCK delays gastric emptying and regulates intestinal content by decreasing gastric activity. A similar action of CCK has been observed in the chicken gizzard (Savory et al, 1981; Martinez et al, 1993). The presence in the distal small intestine of some diet components, most likely lipids, as demonstrated by functional studies (Mateos and Sell, 1981; Mateos et al, 1982; Martinez et al, 1992), would give CCK a physiological function, delaying gastric emptying when the concentration of such components at this level would indicate an overload of the intestine for appropriate digestion in the short transit time through the short avian intestine. Liberation of CCK under such circumstances would guarantee the adaptation of gastric emptying to the capacity of the intestine for lipid digestion. Liberation of CCK by nutrients has not been studied in avian species. Some studies demonstrate the correlation of amino acids in the gastrointestinal tract with immunoreactivity to CCK or gastrin in chicken plasma (Yang et al, 1989). However, the study of Yang et al (1989) does not clarify the mechanism of release of CCK, and the antibody used was nonspecific for this peptide so that the authors were likely determining both gastrin and CCK at the same time. Although the mechanism of release of chicken gastrin has not been studied, for the same reasons, it can be inferred that, as in mammals, amino acids are probably inducing mostly gastrin secretion. This study demonstrates the presence of chicken gastrin in the antrum and of a CCK-like peptide in the intestine, with the highest concentration for the latter in the proximal ileum. These results are in accord with functional studies that also For this reason, the results reported by corroborate the existence of two different Polak et al (1974) and by Shulkes et al. responses to gastrin and CCK in the (1983) stating that endocrine cells and gastrointestinal tract of the chicken.

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Cholecystokinin stimulates intestinal motility whereas chicken gastrin inhibits it. This indirectly demonstrates the existence of two different receptors, one specific for CCK-8 and another for chicken gastrin, further supporting that these two peptides also have different functions in the avian gastrointestinal tract. The fact that the CCK-like peptide present in the intestine reacts with A-CCK in the presence of CCK-8 and also of chicken gastrin-36 indicates that: 1) molecular forms longer that CCK-8 are present in the intestine; and 2) the peptide present in the intestine is different from chicken gastrin. In this sense, isolation and identification studies to determine molecular forms of CCK in avian intestine are needed. The octapeptide of CCK has been identified in the brain of turkeys and chickens (Dockray, 1979; Fan et al, 1987) and this, together with the current results, suggests that whereas CCK-8 is the major molecular form in the central nervous system, longer forms are present in the intestine, a situation similar to that described in mammals (Rehfeld, 1989). To the authors' knowledge there is only one study in which cells containing gastrin have been quantified in avian species (Yamada et al, 1979). An antibody that recognizes both gastrin and CCK was used. As in the Japanese quail, the highest density of immunoreactive cells was found in the antrum and the most dense area occurring in the intestine was the area around the vitelline diverticulum. The reason for the higher density of endocrine cells containing chicken gastrin in comparison with the concentration of G cells in the mammalian antrum (Solcia et al, 1967) could be related to the fact that the so-called antrum in the chicken is a very small area, less than .5 cm2. The gizzard is covered by a keratin-like layer, making it impossible that the mucosa of this organ could be sensitive to the chemical factors of the diet. Endocrine cells were not observed in this area. Thus, all the gastrin cells must be concentrated in the small area of transition between the gizzard and the duodenum.

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ACKNOWLEDGMENTS

The authors are grateful to G. Dockray from the Department of Physiology of the University of Liverpool (P.O. Box 147, Liverpool L69 3BX, UK) for the generous donation of Antisera L48 and L293. This work has been supported by grants from the Universidad Aut6noma de Barcelona and the Direcci6n General de Investigaci6n Cientifica y Tecnica (PB89-0307). The authors are indebted to J. Puertas for his skillful technical assistance.

Coons, A. H., 1956. Histochemistry with labelled antibody. Int. Rev. Cytol. 5:1-23. Dimaline, R., and C. M. Lee, 1990. Chicken gastrin: a member of the gastrin/CCK family with novel structure-activity relationships. Am. J. Physiol. 259:G882-G888. Dimaline, R., J. Young, and H. Gregory, 1986. Isolation from chicken antrum and primary amino acids sequence of a novel 36-residue peptide of the gastrin/CCK family. Fed. Exp. Biol. Sci. Lett. 205:318-322. Dockray, G. J., 1979. Cholecystokinin-like peptides in avian brain and gut. Experientia 35:628-630. Dockray, G. J., 1988. Evolutionary aspects of gastrointestinal hormones. Adv. Metab. Disord. 11: 85-111. Dockray, G. J., and R. A. Gregory, 1989. Gastrin. Pages 311-336 in: Handbook of Physiology, Section 6. The Gastrointestinal System. Vol. n Neural and Endocrine Physiology. S. G. Schultz, G. M. Makhlouf, and B. B. Rauner, ed. American Physiological Society, Bethesda, MD. Fan, Z.-W., J. Eng, M. Miedel, J. D. Hulmes, Y.-C.E. Pan, and R. S. Yallow, 1987. Cholecystokinin octapeptides purified from chinchilla and chicken brain. Brain Res. Bull. 18:757-760. Fumess, J. B., M. Costa, and J. R. Keast, 1984. Choline acetyltransferase and peptide immunoreactivity of submucous neurons in the small intestine of the guinea-pig. Cell. Tissue Res. 237:329-336. Larsson, L.-I., and J. F. Rehfeld, 1977. Evidence for a common evolutionary origin of gastrin and CCK. Nature 269:335-338. Mateos, G. E., and J. L. Sell, 1981. Influence of fat and carbohydrate source on rate of fat passage of semipurified diets for laying hens. Poultry Sci. 60:2114-2119. Mateos, G. E., J. L. Sell, and J. A. Eastwood, 1982. Rate of food passage (transit time) as influenced

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REFERENCES

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