Histochemistry 56, 13-35 (1978)

Histochemistry 9 by Springer-Verlag 1978

Immunoreactivity of the Endocrine Pancreas. Evidence for the Presence of CholecystokininPancreozymin within the A-cell * Dietrich Grube, Volker Maier, Sotirios Raptis, and Werner Schlegel Department of Anatomy and Department of Internal Medicine, Endocrinology and Metabolism, University of Ulm, D-7900 Ulm/Donau, Federal Republic of Germany

Summary. The pancreas of man and rat were investigated immunohistochemically on occurrence and distribution of the "classical" enterohormones gastrin, secretin and cholecystokinin-pancreozymin (CCK-PZ). Concomitantly insulin-, glucagon- and somatostatin-immunoreactive cells have been demonstrated. The unlabelled antibody-enzyme (PAP) method was the most specific and sensitive one when compared with the other immunohistochemical methods performed in this investigation (immunofluorescence, immunoperoxidase). The PAP method reveals reliable and reproducible results, running with high dilutions of antisera and showing a minimum of nonspecific background staining. Extended specificity controls are necessary in immunohistochemistry, however, to exclude nonspecific or crossreactive staining results. F r o m the numerous specificity methods tested, we propose the use of sequential immunohistochemical staining with increasing dilutions of the antisera as an essential control. The use of specific antisera, previously " a d s o r b e d " with related or corresponding antigens (hormones) as Specificity tests should be regarded with some restrictions, at least in the PAP method. When possible a radioimmunological assay should be performed as a final p r o o f of immunohistochemical f i n d i n g s . - T h e immunohistochemical findings of the present investigations have confirmed previous results of other authors concerning occurrence and distribution of insulin-, glucagon- and somatostatin-immunoreactive cells in the pancreas of rat and man. F r o m the enterohormones gastrin, secretin and C C K - P Z only C C K - P Z (or a CCK-PZ-like peptide) was found to occur in the human and rat pancreas. The presence of C C K - P Z in the pancreas was also confirmed by radioimmunological estimations of C C K - P Z in the rat pancreas. The concentration of CCK-PZ-immunoreactive substance was about 1400 pg/g pancreatic tissue (wet weight) and about 1200 pg/g tissue in the small intestine of rats. Concerning the type of endocrine cells in the endocrine pancreas which contain CCK-PZ, it could be demonstrated that obviously the A-(glucagon) cells are immunoreactive to Supported by a grant of the German Research Foundation, SFB 87, Ulm, FRG 0301-5564/78/0056/0013/$04.60

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D. Grebe et al. CCK-PZ. Thus A-cells may contain two polypeptide hormones of different molecular structure. These findings, in addition to other consequences of our results put the " o n e c e l l - o n e hormone theory" in q u e s t i o n . - Gastrinand secretin-immunoreactivity was also present in the A-cells. The immunohistochemical staining was only possible with the PAP method, however. These immunoreactivities in the endocrine pancreas have to be regarded as nonspecific ones, as proved by specificity controls. The secretin-immunoreactivity may be interpreted as a cross-reaction to the structurally similar glucagon. The gastrin-immunoreactivity might be a cross-reaction to the structurally similar CCK-PZ. In fact these cross-reactivities could only be observed in immunohistochemistry but not in the r a d i o i m m u n o a s s a y . - The occurrence of CCK-PZ, a classical enterohormone, in its "target o r g a n " itself forces to reflections on the concept of endocrine and paracrine functions of entero-endocrine cells. Moreover, the functional unity of the endocrine pancreas and the gastrointestinal endocrine system on the one hand as well as the close interrelationships between the endocrine and the exocrine part of the pancreas on the other is emphasized by these findings.

Introduction

For a long period of time the endocrine pancreas and the gastrointestinal endocrine system have been considered as separate endocrine systems, more or less independent from each other. An increasing number of data obtained in various fields of gastrointestinal endocrinology, gave reason for the suggestion that these two systems are a functional unit (cf. Fujita, 1973, 1976, ref.). This hypothesis has been greatly supported by the fact, that some polypeptide hormones coexist in the pancreas and in the gastrointestinal epithelia, e.g. glucagon and glucagon-like immunoreactivity=GLI (see Larsson et al., 1975; Ito and Kobayashi, 1976; Unger et al., 1976; Moody et al., 1977), somatostatin (see H6kfelt et al., 1975; Parsons et al., 1976) and vasoactive intestinal polypeptide=VIP (see Buffa et al., 1977). Little is known, however, about the distribution of the "classical" gastrointestinal hormones (gastrin, secretin, cholecystokinin-pancreozymin = CCK-PZ) within the pancreas as to yet (recent reviews on enterohormones: Barrington and Dockray, 1976; Dawson, 1976; Rayford et al., 1976a, b; Bloom, 1977; Pearse et al., 1977). A lot of contradictory results have been published for instance on the existence of gastrin and gastrin-cells in the normal pancreas and it is still unclear whether this hormone is regularly present in pancreatic tissue (cf. McGuigan, 1972; Lotstra et al., 1974; Braaten et al., 1976; Erlandsen et al., 1976; Dockray et al., 1977). Concerning the occurrence of secretin within the pancreas only one short communication has been published, dealing on secretin-cells in tissue culture of neonatal rat pancreas (Rufener et al., 1976). Investigations on the presence of CCK-PZ in the pancreas as to yet have shown negative results (Polak et al., 1975; Buffa et al., 1976; Dubois et al., 1976). One has to consider, however, that there have been and still are great difficulties in the isolation of pure cholecystokinin, in generating specific antibodies against CCK (without cross-reactivity to the structurally related gas-

Immunoreactivity of the Endocrine Pancreas

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trin) and in the radioactive labelling of CCK for the radioimmunoassay (cf. Harvey, 1974, 1976). We succeeded in raising specific antibodies against naturally occuring CCK-PZ (Mutt, 1976) and in the establishment of a highly specific radioimmunoassay (RIA) for CCK-PZ (Schlegel and Raptis, 1976; Schlegel et al., 1977) and are going now to investigate systematically the distribution of CCK-PZ in the body. The aim of the present investigations was mainly to elucidate the following two problems : 1. Are the classical enterohormones gastrin, secretin and CCK-PZ to be found in the pancreas? (This part of our investigations was completed by including the known islet hormones insulin, glucagon and somatostatin.) 2. Are there differences in the immunohistochemical techniques (immunofluorescence, immunoperoxidase and unlabelled antibody-enzyme (PAP) method) and how are the optimal conditions for gastrointestinal hormones regarding specificity and sensitivity? These studies also discuss the questions which control tests (besides RIA) are valid enough to check the specificity of immunohistochemical reactions. Specificity tests are of particular interest since immunohistochemistry-in contrast to RIA - has no "built-in" assurance of specificity (for details see Sternberger, 1974; Petrusz et al., 1976, 1977; Swaab et al., 1977).

Material and Methods l. Tissue Preparation Samples of human pancreas (surgical specimens or obtained immediately after death) and rat pancreas (Sprague-Dawley, Ivanovas, Kisslegg, FRG) were fixed according to the following procedures: Bouin's fluid (with and without acetic acid), 3-12 h; 4-10% formaldehyde solution, I2-24 h; vapor phase fixation with paraformaldehyde after freeze-drying according to Falck et al. (1962); vapor phase fixation with diethylpyrocarbonate (DEPC) after freeze-drying according to Pearse et al. (1974).-After fixation the tissue was embedded in paraplast (in vacuo after freeze-drying). Tissue sections were cut at 5 gm and fixed on albuminized slides. The sections were incubated in air in an incubator at + 3 7 ~ for at least 48 h. Subsequently the sections were rehydrated after removal of the paraffin, rinsed in destilled water and 0.01 M phosphate-buffered saline (PBS) of pH 7.3 and processed for immunohistochemistry.

2. Antibody Preparation Antisera against 99% purified naturally occurring CCK-PZ (Prof. V. Mutt, Stockholm, Sweden), highly purified naturally occurring secretin (Prof. V. Mutt) and pure synthetic secretin (Prof. E. Wtinsch, Miinchen, FRG), purified porcine glucagon (Novo, Bagsvaerd, Denmark) and synthetic gastrin-ns (Imperial Chemical Ind., Macclesfield, Great Britain) were raised irl rabbits by coupling these peptides with N,N'-carbonyldiimidazole to bovine serum albumin (for details see Schlegel and Raptis, 1976). None of these antisera showed any cross-reactions with gut and pancreatic hormones tested for cross-reactivity (insulin, pancreatic glucagon, natural 99% purified secretin, synthetic secretin, gastrin (G 17-ns), motilin, gastric inhibitory polypeptide (GIP) and vasoactive intestinal polypeptide (VIP)).-Somatostatin-antisera were obtained from Prof. A, Arimura, New Orleans, USA,-Guinea-pig antisera against bovine insulin were obtained from Miles Res. Products, Elkhart, USA.

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3. Immunohistochemistry The following immunohistochemical methods were mainly performed for the studies in h u m a n and rat pancreas. As controls to the results from the pancreas immunohistochemistry was also performed with samples from h u m a n and rat stomach and small intestine.

a) Immunofluorescence. According to Coons et al. (1955) an indirect immunofluorescence technique was applied: antisera against glucagon, insulin, CCK-PZ, gastrin and secretin served as first layers. The antisera were diluted 1:10, 1:20, 1:30, 1:50 and 1:100 in 0.01 M PBS at pH 7.3. The second layer was FITC-conjugated swine anti-rabbit serum IgG (Dakopatts, Denmark), diluted l : 10, 1 : 20, 1:30, 1:50 and 1:100 in PBS. Incubation times (room temperature) for the antisera and IgG were one hour. The antisera were treated with 1% normal swine serum and bovine serum albumin (1 mg/ml) to avoid nonspecific background staining. After each incubation step all sections were rinsed in PBS containing 0.1 M saccharose for one hour. Subsequently the sections were mounted in PBS-buffered glycerine and observed in'a Leitz Orthoplan | microscope equipped with a surface illumination system and an Orthomat | camera, To prove the specificity of the immunofluorescence some sections were incubated with normal (nonimmunized) rabbit serum instead of specific antisera or single steps of the immunohistochemical procedure have been omitted or replaced with PBS. b) Immunoperoxidase Method. This technique was performed on principle according to Piris and Whitehead (1974) for the immunohistochemical proof of one single hormone and according to Nakane (1968) for the simultaneous localization of two different hormones in the same s e c t i o n . - Antisera against glucagon, insulin, CCK-PZ, gastrin and secretin in various dilutions (see i m m u n o fluorescence) served as first layers. The second layer was peroxidase-conjugated swine antirabbit serum IgG (Dakopatts, Denmark), diluted as described before. Peroxidase " s t a i n i n g " was performed either according to G r a h a m and Karnowsky (1966) with 3,3'-diaminobenzidine (DAB; 12.5 rag/ 100 ml 0.05 M Tris-HCl-buffer at p H 7.6 plus 0.4 ml 1% H~O2) or with 4-chloro-1-naphthol according to Nakane (1968). Following peroxidase staining some sections have been incubated for ten minutes in vapor phase of OsO4 (100 mg/1 1 glass jar). Subsequently the sections were mounted in PBS, PBS-buffered glycerine, PVP or in Eukitt (after dehydration in graded alcohols and clearance in X y l o l ) . - The specificity-controls for the immunohistochemical reactions were: incubation of the sections with normal rabbit serum instead of antisera; incubation of the sections in unlabelled swine anti-rabbit serum IgG before the incubation in peroxidase-labelled swine anti-rabbit serum IgG; omission of single steps or replacement by PBS in the immunohistochemical procedure, For the identification of two hormones in the same tissue section, the first hormone was localized as described before, using either DAB (brown) or 0.04% 4-chloro-l-naphthol (bluegrey) as reaction product (incubation times: 3, 5, 8, 11 and 15 min). Subsequently the sections were immersed for one hour in glycine-HCl-buffer at p H 2.2. After a short rinse in PBS the second h o r m o n e was demonstrated by incubation in the second antiserum and in peroxidase-labelted swine anti-rabbit serum IgG. Depending on the staining reaction of the first immunoreaction, peroxidasestaining was performed either with D A B or with 4-chloro-l-naphthol (incubation times as above). T h e " double-stained" sections were m o u n t e d (due to the solubility of 4-chloro- 1-naphthol in organic solvents) only in PBS-buffered g l y c e r i n e . - T h e following combinations of antisera have been tested in the double-staining method: gastrin - CCK-PZ, gastrin - secretin, C C K - P Z - secretin, C C K - P Z gastrin, s e c r e t i n - gastrin, secretin - CCK-PZ, C C K - P Z - glucagon, glucagon - CCK-PZ, glucagon insulin, i n s u l i n - g l u c a g o n . Specificity controls for the sequential immunohistochemical staining methods were accomplished according to Nakane (1968).

c) Unlabelled Antibody-Enzyme (PAP) Method. In the PAP procedure tissue sections have been treated (with slight alterations) according to the techniques as published by Sternberger (1974): deparaffinized, rehydrated sections were rinsed in 0.01 M PBS at pH 7.3 and incubated at + 4 ~ C for 38~,8 h with one of the following antisera: anti-insulin, anti-glucagon, anti-somatostatin, antiC C K - P Z , anti-gastrin, and anti-secretin. Working dilutions in PBS at pH 7.3 of the antisera were: 1:10, 1:100, 1:200, 1:300, 1:500, 1:1000, 1:1500, 1:2000 and in some cases also 1:5000 and 1:10000. In addition sections have been treated, instead of specific antisera, with a serum of a non-immunized rabbit (showing nevertheless a special kind of immunoreactivity, see below)

Immunoreactivity of the Endocrine Pancreas

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at the same working dilutions.-After incubation in the antisera the sections were shortly rinsed in PBS and incubated for ten minutes in non-labelled swine anti-rabbit serum IgG (Dakopatts, Denmark), diluted 1 :i0 in 0.01 M PBS at pH 7.3. After another rinse in PBS the sections were treated with the peroxidase-anti-peroxidase (PAP) complex (Dakopatts, Denmark), diluted i:50 in PBS for ten minutes. Staining of peroxidase was performed, as described before, for ten minutes. Finally, the sections were osmicated at room temperature with vapor phase of OsO4 for ten minutes. The sections were mounted without counterstaining in PBS, PBS-buffered glycerine, PVP or (after dehydration) in Eukitt. -- The less time consuming PAP procedure as published by Burns (1975) was also performed. For systematic investigations the above described procedure was found to be more useful. - Blocking of the endogen peroxidase, performed with various methods (cf. Taylor, I974; Burns, 1975; Reading, 1977) was found to influence the immunoreactivity of gastrointestinal and pancreatic endocrine cells. Therefore the tissue sections were processed without blocking of the endogen peroxidase during these investigations. - In order to identify a presumptive content of two different hormones in the same cell type, adjacent tissue sections were exposed to the following sequences of antisera: anti-glucagon-anti-gastrin-anti-somatostatin-anti-insulin; antglucagon - a n t i - C C K - P Z - anti-somatostatin - anti-insulin; anti-glucagon - anti-secretin - anti-som a t o s t a t i n - a n t i - i n s n l i n . - I n order to test the reduction of nonspecific background staining one of the following substances were added to the antisera and buffer solutions respectively: normal (non-immune) rabbit serum, normal swine serum, bovine serum albumin and human serum albumin.-Specificity of the immunohistochemicaI reactions in the PAP-procedure was proofed by: incubation of sections with sera of non-immunized rabbits instead of specific antisera; omission of single steps or replacement with PBS during the PAP procedure; use of specific antisera which have been "blocked" by adsorption with an excess of purified corresponding antigen; incubation of sections in antisera previously adsorbed with related or unrelated antigens; sequential staining of adjacent sections with increasing dilutions of a n t i s e r a . - T h e sections were examined under a Leitz Orthoplan | microscope, photomicrographs were taken with a Leitz Orthomat | camera.

4. Radioimmunoassay a) For CCK-PZ: tissue samples of the pancreas (divided into caput, corpus and cauda pancreatis), duodenum and jejunum of male rats were dissected free immediately after killing the animals. Tissue preparation and the radioimmunological determinations of CCK-PZ were carried out following the method of Schlegel etal. (1977). For the specificity of the assay cross-reactivity studies were performed with gastrin, motilin, GIP, synthetic secretin, naturally occurring secretin, insulin, pancreatic glucagon and VIP. No cross-reactivity with all these polypeptides - i n c l u d i n g gastrin (!) couid be detected.

b) For Glucagon: the serum of a rabbit, which never had been immunized by any polypeptide hormone, showed immunoreactivity in the endocrine pancreas (see below). This serum was radioimmunologically assayed for glucagon. The RIA was performed by a modification of the method of Heding and Rasmussen (1972) in 0.08 M veronal buffer, using 125I-monoiodinated glucagon (182gCi/gg; New England Nuclear). After 48h incubation the free hormone was separated from the antigen-antibody complex by the addition of dextran-coated charcoal (Herbert etal., 1965). Twice crystallized pork glucagon (Novo) was used as standard.

Results

1. Tissue Processing and Immunohistochemical Techniques a) Fixation. A l l o f t h e t e s t e d f i x a t i v e s w e r e m o r e o r less s u i t a b l e f o r i m m u n o h i s t o c h e m i c a l i n v e s t i g a t i o n s (see a l s o A r n o l d Best results, however, were obtained after

e t al., 1 9 7 5 ; B o s m a n e t al., 1977). fixation of the tissue in Bouin's

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fluid (cf. Erlandsen et al., 1976) and there have been no substantial differences between Bouin's fluid with and without addition of acetic acid. A short fixation time is recommended (1-3 h), but parenchymatous organs like the pancreas should be fixed for at least 6 h. - Fixation of the tissue in formaldehyde (liquid, or vapor phase after freeze-drying) is also a valid method in immunohistochemistry, even though in tissues stored for a longer period of time a relatively high background fluorescence appears in the immunofluorescence m e t h o d . The vapor phase fixation of the tissue in DEPC (Pearse et al., 1974) in our own experiences proofed not to be very useful. There is only poor preservation of the structure. Moreover, background staining was relatively strong and there were no reliable or reproducible results in immunohistochemical staining. For instance, immunoreactive cells occurred in gastrointestinal epithelia but no immunoreactivity could be seen in the islet cells of the pancreas. Another disadvantage of freeze-dried preparations is that only small tissue blocks can be processed.

b) Immunohistochemical Methods. The most rapid operating immunohistochemical technique is immunofluorescence. Compared to the PAP procedure immunofluorescence is relatively poor sensitive and there are often disturbing autofluorescent structures inthe tissue. For special purposes, however, e.g. the simultaneous demonstration of amine-containing and polypeptide-containing endocrine cells, this method is very useful (cf. Forssmann et al., 1976; Grube et al., 1977). FITC-fluorescence fades during examination under ultraviolet light, documentation of results is therefore somehow difficult. A further disadvantage of immunofluorescence (and the immunoperoxidase method) is the relatively high consumption of antisera. The highest dilution of antisera for getting reliable results with these techniques in the field of gastrointestinal polypeptide hormones was in our investigations a dilution rate of about 1 : 4 0 . - T h e unlabelled antibodyenzyme (PAP) technique is a method including the most processing steps in immunohistochemical staining. Compared with the other tested techniques, however, the PAP t e c h n i q u e - a s confirmed also in our investigations- has the highest sensitivity (cf. Burns, 1975). Background staining is faint, even if sections are postosmicated by vapor phase OsO4. The dilution of antisera used in this method should be at least about 1:200. Higher concentrated antisera often reveal a strong background staining and even a nonspecific or lacking staining of endocrine cells (!). On the other hand even an antiserum-dilution up to 1:10000 reveals specific immunohistochemical staining. For reproducible results antisera-dilution in the range of 1 : 1 0 0 0 - 1 : 1500 is recommended. e) Specificity Controls. When single steps of the immunohistochemical procedure had been omitted or replaced by PBS, no immunohistochemical staining could be observed. The same results have been found in controls where specific antisera had been replaced by sera of non-immunized animals. One exception occurred, because one of our " n o r m a l " rabbit sera showed specific staining of A-cells in the pancreatic islets (Fig. 6). The reaction only could be seen using the PAP method, neither the immunofluorescence nor the immunoperoxidase method showed staining in this case. Moreover, the immunoreactivity of this serum against A-cells was limited to dilutions of the antiserum up to 1:400.

Immunoreactivity of the Endocrine Pancreas

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Though the rabbit from which this serum was obtained had never been immunized with any polypeptide hormone, a subsequent RIA detected a glucagonbinding capacity of this serum of 16% (nonspecific binding in this assay was 4%). This would suggest that also sera of non-immunized animals may contain (auto ?-) antibodies against polypeptide hormones. When replacement of specific antisera by " n o r m a l " sera is performed as a specificity test in immunohistochemistry this fact should be kept in mind (cf. Sternberger, 1974; Petrusz et al., 1976; Swaab et al., 1977). A rather reliable specificity test, mainly in the sensitive PAP procedure, based on our experiences, are simultaneous staining reactions with "increasing dilutions of the primary antiserum. With this test system the immunoreactivity of pancreatic endocrine cells to secretin- or gastrin-antisera could be ruled out as nonspecific reactions in the present investigations (see below). - The use of " a d s o r b e d " ("saturated") antisera is the most widely used specificity test in immunohistochemistry. Our investigations, however, have shown that this type of specificity control has to be considered with some restrictions (see also Swaab etal., 1977). Apart from non-expected " a d s o r p t i o n s " (see below), some technical data in the processing of saturated antisera are of critical importance. The adsorption of antisera with antigens needs at least 36-48 h. The storage of antisera at room temperature or even at + 4 ~ (without addition of pure hormones) may reduce the immunoreactivity of the antisera to a certain degree, particularly the immunoreactivity of our CCK-PZ-antiserum. This fact may lead to misinterpretations of adsorption tests. Therefore adsorption studies in immunohistochemical techniques should be performed with antisera-dilutions of about 1 : 300 1 : 500 (valid for the PAP method), where not adsorbed antisera (incubated as controls in appropriate solving media of the antigen) remain in any case reactive. The amount of antigen, necessary for the saturation of an antiserum, varies from one antiserum to the other." Excess" amounts of antigen (10 btg-100 btg/ml serum) are necessary for a complete " a d s o r p t i o n " of the immunoreactivity of specific antisera. As discussed below, the addition of such excess amounts of antigen to antisera may lead to changes of the binding capacity of antigens and antibodies respectively, even if " u n r e l a t e d " antigens are added to specific antisera. Special problems may occur with the solving media used for the solution of pure hormones, particularly in those cases where acid pH is necessary for the solution of the antigens (e.g. gastrin and glucagon). Control studies revealed that, even if the final solution (solving medium of the antigen plus antiserum) is adjusted to pH 7.3, antisera may become unreactive, also without addition of antigens. In these cases we dissolved the pure hormones in 0.1-0.0I N HC1 diluted with Tris. An occasionally appearing turbidity could be removed by a short ultrasonic treatment. Subsequently the antiserum was added to the solution in appropriate amounts.

d) Background Staining. Reduction of background staining in immunohistochemistry can be achieved by addition of normal sera to the antisera. In our investigations the addition of 1% normal rabbit serum to the antisera

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(raised in rabbits) and the addition of 1% normal swine serum to anti-rabbit serum IgG (raised in swine) reduced background staining in the PAP method satisfactory. To prevent the staining of connective tissue fibers, the addition of human serum albumin (1 mg/ml) was found most useful.

2. Immunoreactivity of the Endocrine Pancreas a) Insulin- and glucagon-immunoreactive cells were demonstrated in the pancreas of rat and man with all above mentioned immunohistochemical methods. Concerning number and distribution pattern of these cell types the findings of other authors could be confirmed (cf. Orci et al.,1976b; Erlandsen et al., 1976; reviews: Epple and Lewis, 1973; Lange, 1973). In the rat insulin-immunoreactive cells (the most frequent endocrine cell types) are situated in the center of the islets (Fig. 9d), surrounded by a " m a n t l e " of glucagon-immunoreactive cells (Fig. 9b). The number of the latter cells per islet varies in different parts of the rat pancreas. In the uncinate process there are e.g. relatively few glucagon cells. In this part of the pancreas the mantle of the islet consists mainly in pancreatic polypeptide-(PP) cells (cf. Erlandsen et al., 1976; Orci et al., 1976 a; Sundler et al., 1977). In the human pancreas glucagon-immunoreactive cells are more or less irregularly distributed within the islets (Fig. 8a). They are less frequent than insulin-immunoreactive cells. b) Somatostatin-immunoreactive cells are present in the human and rat pancreas as well as in the gastrointestinal epithelia. In the rat pancreas this cell type is encountered relatively seldom (Fig. 9c). Often, but not regularly, the somatostatin-cells show an interposition between insulin- und glucagon-cells, as stressed by Unger and Orci (1977). In the human pancreas somatostatin-immunoreactive cells are more frequent than in the pancreas of the rat. Moreover in the human pancreas single somatostatin-cells can be seen intercalated between exocrine cells. c) CCK-PZ-immunoreactive cells could be observed with all immunohistochemical techniques used in these investigations, and t h a t - a p a r t from the intestinal e p i t h e l i a - i n the human and rat endocrine pancreas. Following immunofluorescence (Fig. 1) as well as with the immunoperoxidase and the PAP method (Figs. 2, 3, 9a), CCK-PZ-immunoreactive cells were found regularly in the islets. Concerning number and distribution pattern, these cells most probably correspond to the (glucagon-containing) A-cells. In the rat pancreas CCK-PZ-immunoreactive cells are situated peripherally in the islets (Figs. 1, 2, 3, 9a). In the human pancreas they are more or less irregularly scattered between the other islet cells (Fig. 8b). d) Gastrin- and secretin-immunoreactive cells could not be detected by the immunofluorescence and immunoperoxidase method in human as well as in rat pancreas, though ~these methods were suitable for the demonstration of gastrinand secretin-cells in the gastrointestinal epithelia. Using the PAP method, how-

Immunoreactivity of the Endocrine Pancreas

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Fig. l a and b. Immunofluorescent staining of CCK-PZ in the pancreas of the rat, CCK-PZ-immunoreactive cells are arranged peripherally in the islets, a An islet from the body of the pancreas. x 320. b An islet from the uncinate process of the pancreas, x 320 Fig. 2, Duodenum and adjacent part of the rat pancreas. CCK-PZ-immunoreactivity (PAP-method, a n t i s e r u m - d i l u t i o n 1:1000) in an islet of Langerhans and in a cell of a duodenal gland (arrow). xlT0

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ever, gastrin- and secretin-immunoreactivity occurred also in the pancreas, and that most probably in A-cells (Figs. 4, 5). In contrast to the CCK-PZ-immunoreactivity, the immunoreactivity of gastrin and secretin in the endocrine pancreas was only present up to a antiserum-dilution of about 1:500 (see below).

e) Specificity of CCK-PZ-, secretin- and gastrin-immunoreactivity in the endocrine pancreas. Apart from radioimmunological estimations of CCK-PZ, extended specificity controls (see material and methods) of the results obtained with the PAP method have been performed. From these studies the following results should be pointed out. Use of increasing dilutions of antisera: the gastrin-, secretin- and CCK-PZantisera have been diluted in increasing dilutions from 1 : 10 to 1 : 1500, in part also to 1:5000 and 1:10000. It was remarkable to note that a specific immunohistochemical staining of entero-endocrine and pancreatic endocrine cells was absent if antisera in a dilution of 1:10 to 1:100 were used in the PAP method. Concerning the pancreas, the islet cells either were unreactive or the entire islets were stained irregularly. Usually a strong background staining was visible. On the other hand a clearly visible immun6reactivity against anti-gastrin, antisecretin and anti-CCK-PZ occurred in peripherally located islet cells (A-cells), using antisera-dilutions of about 1:200. Differences in the staining intensity between these antisera, however, are to be seen (Figs. 3, 4, 5). These differences became more pronounced with further increasing dilutions of the antisera, which abolished stepwise the staining of A-cells with secretin- and gastrin-antisera (Fig. 4). At a dilution of 1:1500, from the three antisera only CCK-PZ-antisera revealed immunoreactivity of the A-cells (Fig. 3c), whereas antisera against gastrin and secretin at this dilution were ineffective in the endocrine pancreas (but still effective in the gastrointestinal gastrin- and secretin-cells). Thus the CCK-PZ-antiserum was comparable with the other antisera against islet hormones (glucagon, insulin, somatostatin), which also could be used at dilutions of 1:1500 and higher for the immunohistochemical staining of specific islet cells. Use of "adsorbed" antisera: the immunoreactive staining of the A-cells with gastrin- and secretin-antisera was relatively weak. Specificity tests of this weak immunoreaction were not useful, because of the above mentioned reasons. Therefore systematic adsorption studies (immunohistochemical staining with the PAP method) were only performed with the CCK-PZ- and the glucagon-antisera and the pure hormones glucagon, gastrin and CCK-PZ. Incubation of the CCKPZ-antiserum with highly purified CCK-PZ (at least 50 btg/ml) diminished or abolished - a c c o r d i n g to the amount of added pure CCK-PZ - t h e immunoreactivity of the A-cells in the pancreatic islets (Fig. 7) as well as the immunoreaction of gastrointestinal CCK-PZ-cells. No " s a t u r a t i o n " however of the CCK-PZantiserum occurred by the addition of gastrin, a polypeptide structurally related to CCK-PZ. Surprisingly such a saturation of the CCK-PZ-antiserum took place also with glucagon (Novo), a potypeptide of a totally different amino-acidsequence than CCK-PZ. We consider this, however, not as a "specific" adsorption in the sense of an antigen-antibody-reaction, as will be discussed b e l o w . The glucagon-antiserum could be saturated with pure glucagon (20 gg/ml), but

Immunoreactivity of the Endocrine Pancreas

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Fig. 3a-c. CCK-PZ-immunoreactivity (PAP method), using varying dilutions of antisera, in the endocrine pancreas of the rat. a Antiserum-dilution 1:200. x 260. b Antiserum-dilution 1:500. x 260. e Antiserum-dilution 1 : 1500. x 420

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Fig.4a and b. Secretin-immunoreactivity in the endocrine pancreas of the rat (cf. Fig. 3). a Antiserum-dilution 1:200. x 260. b Antiserum-dilution 1: 1000. x 170

Fig. 5. Gastrin-immunoreactivity in the endocrine pancreas of the rat (cf. Figs. 3 and 4). Antiserumdilution 1:200. x 420 Fig. 6. Immunoreactivity of A-cells in the endocrine pancreas of the rat to a serum of a "nonimmunized" rabbit (PAP method, serum-dilution 1 : 200). x 260

not with CCK-PZ or gastrin.-In a n y case the a m o u n t s o f p u r e h o r m o n e s , essential for t h e s a t u r a t i o n o f specific a n t i s e r a a n d the a b o l i t i o n o f t h e i m m u n o h i s t o c h e m i c a l s t a i n i n g in t h e P A P m e t h o d , a r e h i g h l y b e y o n d t h o s e u s e d in specificity tests o f r a d i o i m m u n o l o g i c a l t e c h n i q u e s . T h e r e f o r e the m e c h a n i s m o f r e a c t i o n s i n v o l v e d in the s a t u r a t i o n o f a n t i s e r a w i t h a n t i g e n s c a n h a r d l y be c o n s i d e r e d as a "simple" a n t i g e n - a n t i b o d y b i n d i n g (cf. discussion).

Fig. 7a and b. CCK-PZ-immunoreactivity in the endocrine pancreas of the rat, using " a d s o r b e d " antisera, a Atatiserum, diluted 1:200 and adsorbed with 20 pg pure CCK-PZ/ml. x 260. b Antiserum, diluted I: 200 and adsorbed with 100 gg pure CCK-PZ/ml. (Islet encircled) x 260

Fig. 8. Immunoreactivity in adjacent sections of the same islet of Langerhans (human pancreas). a Glucagon-immunoreactivity. b CCK-PZ-immunoreactivity. x 260

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Fig. 9a-d. Sequential staining of the same islet of Langerhans in adjacent sections of rat pancreas. a CCK-PZ-immunoreactivity. b Glucagon-immunoreactivity. c Somatostatin-immunoreactivity. d Insulin-immunoreactivity. x 260

f) Identity of glucagon- and CCK-PZ-immunoreactive cells. To p r o v e the identity o f C C K - P Z - a n d g l u c a g o n - c o n t a i n i n g A-cells, serial sections o f h u m a n a n d rat p a n c r e a s were sequentially p r o c e s s e d with C C K - P Z - a n d g l u c a g o n - a n t i s e r a . In a d d i t i o n i m m u n o h i s t o c h e m i c a l d o u b l e staining p r o c e d u r e s ( N a k a n e , 1968) were p e r f o r m e d in the p a n c r e a s (and g a s t r o i n t e s t i n a l epithelia) o f the rat. By

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sequential staining of serial sections, alternatively with glucagon-, CCK-PZ-, somatostatin- and insulin-antisera, there was good evidence for the presumption, that CCK-PZ-immunoreactive cells are identic with the glucagon-immunoreactire A-cells. Because of the distribution pattern of A-cells, this was more striking in the rat pancreas (Fig. 9) than in the human pancreas (Fig. 8 ) . - An additional proof for this identity of glucagon- and CCK-PZ-immunoreactive cells came from investigations of the pancreas in a case of hyperinsulinism (unpublished data). In this pancreas glucagon-immunoreactive cells were confined to the duodenal part of the pancreas, whereas the splenic part of the pancreas was without these cell types. The same distribution pattern could be observed for CCK-PZ-immunoreactive cells. The attribution of CCK-PZ-immunoreactivity to glucagon-cells could also be confirmed by use of an immunohistochemical double staining method, performed in the rat pancreas: in contrast to the results obtained with the antisera-sequences glucagon-insulin or insulin-glucagon, where different endocrine cell types could be demonstrated simultaneously in the same section by this method, only one cell type could be stained in the islets using the antisera-sequence g l u c a g o n - CCK-PZ or C C K - P Z - glucagon. At a certain constellation of reaction times (DAB-staining 3-5rain, 4-chloro-l-naphthol 5-10 min) an ill definable intermediate colour (brown-violet) could be observed in the A-cells. This could mean that CCK-PZ-immunoreactivity is located in the same cells as glucagon-immunoreactivity.

g) Radioimmunoassay. Radioimmunological estimations of CCK-PZ were performed in the small intestine (first part of the duodenum and first part of the jejunum) and in the pancreas (divided into different parts) of the rat. The immunoassayable CCK-PZ was 893 pg/g tissue (wet weight) in the first part of the duodenum and 1382 pg/g tissue in the first part of the jejunum. In the pancreas 1491 pg/g tissue were detected in the head, 1354 pg/g tissue in the body and 1392 pg/g tissue in the t a i l . - I f we consider that the weight of the small intestine is considerably determined by its muscular layer and by connective tissue, whereas the pancreas only contains relatively few connective tissue, the rat pancreas does contain even more CCK-PZ/epithelial structures than the small intestine. (Radioimmunoassay for glucagon in " n o r m a l " rabbit serum see page 19.)

Discussion

The aim of the present investigations firstly was to study the immunoreactivity of the endocrine pancreas with the classical gastrointestinal hormones gastrin, secretin and CCK-PZ. In addition the glucagon-, insulin- and somatostatinimmunoreactive cells were investigated. Different immunohistochemical methods and specificity tests in immunohistochemistry have been c o m p a r e d . - Regarding the presence and distribution of insulin-, glucagon- and somatostatin-immunoreactive ceils in the pancreas of man and rat, our findings agree with those of other authors (e.g. Erlandsen et al., 1976; Orci et al., 1976b). The most essential result of our investigations, however, is the immunohistochemical and

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radioimmunological proof of CCK-PZ in the endocrine pancreas, detected for the first time in this location. From all the questions resulting from these findings we will focus within this discussion on three problems. 1. Specificity of the CCK-PZ-immunoreactivity. 2. CCK-PZ-immunoreactivity and pancreatic A-cells. 3. CCK-PZ and the gastro-entero-pancreatic endocrine system.

Specificity of CCK-PZ-Immunoreactivity Compared with other gastrointestinal or pancreatic polypeptide hormones (e.g. gastrin or glucagon), the generation of antibodies against CCK-PZ and its radioimmunological determination have proved to be very difficult. This is mainly due to the relative scarcity of pure hormone, its poor immunogenity and the instability of most radioactively labelled preparations. Moreover cross reactions of CCK-PZ-antisera with the structurally similar polypeptide gastrin could hardly b e avoided (cf. Harvey, 1974). We succeeded in the preparation of CCK-PZ-antibodies, showing no cross-reactivity with gastrin or other enteric or pancreatic hormones, and in the establishment of a specific RIA for CCK-PZ (Schlegel and Raptis, 1976; Schlegel et al., 1977). Apart from gastrointestinal epithelia, we now have evidence for the regular occurrence of relatively high amounts of CCK-PZ in the pancreas of rat and man. This is in contrast with findings of other authors (Polak et al., 1975; Buffa et al., 1976; Dubois et al., 1976). These controversial results may partly be due to differences between our antibodies and those of other laboratories, since informations on presumptive molecular variants of CCK-PZ and the immunogenic determinants are lacking. In any case our CCK-PZ-antisera as well as our RIA for CCK-PZ are highly specific (cf. Schlegel et al., 1977).-However, in contrast to RIA, which shows an " i n b o r n " specificity, immunohistochemical techniques are of no predetermined specificity (see Sternberger, 1974; Petrusz et al., 1976, 1977; Swaab et al., 1977). Therefore extended specificity tests in histochemistry have been necessary during our investigations. All these t e s t s - t h e use of " s a t u r a t e d " antisera with some restrictions, see b e l o w - s h o w e d that the proof of CCK-PZimmunoreactive cells in the endocrine pancreas has to be regarded as specific. In this connection, the use of increasing dilutions of antisera in the unlabelled antibody enzyme (PAP) method obviously is of special significance. This method has also confirmed that the PAP method is much more sensitive than other immunohistochemical techniques like immunofluorescence and immunoperoxidase methods. The PAP staining shows immunoreactions with high-diluted antisera and even traces of "cross-reactivities" between specific antisera and related antigens can be detected (what vice versa also could be considered as a disadvantage). The use of increasing dilutions of antisera, however, offers a rather good discrimination between specific and nonspecific immunohistochemical staining as well as a clue for the titer of an antiserum. This can hardly be ascertained by running appropriate tests in other immunohistochemical techniques. Concerning the immunoreactivity of the endocrine pancreas against anti-gastrin, antisecretin and anti-CCK-PZ, we could show that the CCK-PZ-immunoreactivity

[mmunoreactivity of the Endocrine Pancreas

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(corresponding to the results with anti-insulin, anti-glucagon and anti-somatostatin) is practically unaltered by increasing dilutions of the antiserum. The immunoreactivity of islet cells against anti-gastrin or anti-secretin, however, has to be regarded as nonspecific, since these immunoreactivities were greatly diminished with increasing dilutions of the antisera. In fact, we regard the immunoreactivity of the pancreatic A-cells against antigastrin in our investigations as a cross-reaction against CCK-PZ, structural similar with gastrin and located in the A-cells. The secretin-immunoreactivity of A-cells may be due to a crossreactivity of anti-secretin against glucagon, which is structurally related with secretin (as to the hormonal "families" of gastrointestinal polypeptide hormones, refer to the review given by Barrington and Dockray, 1976). These cross-reactivities were found in immunohistochemistry with antisera, which previously had been shown in RIA to be without any cross-reactivity (Schlegel et al., 1977). Thus, specificity controls, obtained in RIA, are of restricted significance in immunohistochemical techniques and have to be proved in immunohistochemistry by additional specificity t e s t s . - Despite our findings, gastrin- or secretinimmunoreactive cells may occur in the pancreas (Braaten et al., 1976; Erlandsen et al., 1976; Rufener et al., 1976; Dockray et al., 1977). Most likely the gastrinand secretin-antisera of the cited authors have been raised against other molecular forms of these hormones than ours (and e.g. those of Lotstra et al., 1974). We believe, however, that at least some of previous findings concerning the presence of immunoreactive gastrin-cells in the endocrine pancreas can be explained as cross-reactivities of gastrin-antisera (formerly of lower specificity than today) against CCK-PZ, now detected as a regular constituent of the endocrine pancreas. Some remarks are also necessary on the specificity tests using " a d s o r b e d " antisera. Antisera, previously incubated with corresponding or related hormones, are widely used in immunohistochemistry as specificity controls, even though the molecular processes, involved in this reaction are still unknown (cf. Sternberger, 1974; Petrusz et al., 1976, 1977; Swaab et al., 1977). In our present studies, CCK-PZ-antisera, saturated with pure CCK-PZ, showed no immunoreactivity. This test could be regarded therefore as a proof of our findings. On the other hand, the CCK-PZ-antiserum could also be adsorbed (in immunohistochemistry, but not in RIA) with the structurally unrelated polypeptide glucagon, but never w i t h - w h a t would have been more p r o b a b l e - t h e structurally related pure hormone gastrin. This saturation of the CCK-PZ-antiserum with glucagon permits several explanations : firstly, the glucagon (Novo), used in our adsorption studies, can not be regarded as " p u r e " in a biochemical sense. Impurities of this glucagon may contain CCK-PZ, especially since these both hormones obviously are located in the same islet cells. A clarification of this peculiar "cross reactivity" between the CCK-PZ-antiserum and glucagon may perhaps be possible, when highly purified glucagon-preparations are available. - Secondly, it is known from theoretical (Ekins, 1974) as well as from practical standpoints (Burger and Franchimont, 1974) that the addition of "excess" quantities of antigens to specific a n t i s e r a - a s it is usually performed during these kind of specificity t e s t s - i n d u c e s several disturbing factors. These factors influence the binding capacity or binding reactions in a manner, that antisera seem to be " s a t u r a t e d "

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with structurally not related antigens. - After the special events in our adsorption tests, this kind of specificity tests has to be regarded with some restriction in immunohistochemistry-unless an absorption procedure can be established for immunohistochemical purposes, working with amounts of pure hormones, equivalent to saturation studies in the RIA. - Since sections are incubated during the PAP procedure for about 48 h with the saturated antisera, a competitive exchange of added and tissue-bound antigens may take place at the antibody site. Saturation of antisera with pure hormones by solid phase adsorption (preincubation of the antiserum with sepharose to which the antigens are bound, cf. Swaab et al., 1977) would make the adsorption tests more specific.- Despite the " s a t u r a t i o n " of our CCK-PZ-antiserum with glucagon, we are sure to have demonstrated CCK-PZ or a CCK-PZ-Iike immunoreactivity in the endocrine pancreas. In this context it seems not only possible but even probable thai the "pancreatic" C C K - P Z - analogous to e.g. glucagon and glucagon-likeimmunoreactivity (GLI, see Unger et al., 1976) or the different molecular forms of gastrin (cf. Yalow and Straus, 1977)-has another molecular structure than the " e n t e r i c " CCK-PZ (see also Mutt, 1 9 7 6 ) . - T h e immunoreactivity of the pancreatic A-cells against CCK-PZ-antisera could also be due, more theoretically, to a peptide with a identical molecular structure, or to an amino-acid sequence within a peptide, corresponding to the immunogenic determinant of CCK-PZ (e.g. "glicentin", see Moody et al., 1977). Another polypeptide which could be responsible for the CCK-PZ-immunoreactivity in the endocrine pancreas is the "gastrosecretagogue pancreatic peptide" = GSP (Pointner, 1975). This peptide shows a marked stimulatory effect on gastric acid secretion. On the other hand " G S P " may also be nothing else but a fragment of other islet polypeptides, e.g. of glicentin or CCK-PZ.

CCK-PZ-Immunoreactivity and A-Cells Apart from the specificity of CCK-PZ-immunoreactivity in the endocrine pancreas the identity of the CCK-PZ-immunoreactive cells is of special interest. For this identification of CCK-PZ-cells various methods have been used. The exposure of adjacent tissue sections to different sequences of antisera (glucagon, CCK-PZ, somatostatin and insulin) as well as the sequential incubation of the same tissue section in different antisera (immunohistochemical double staining method, see Nakane, 1968) revealed, that the CCK-PZ-immunoreactivity most probably has to be attributed to the A-cells. With respect to the distribution pattern of CCK-PZ-immunoreactive cells within the rat pancreatic islets, theoretically also pancreatic polypeptide (PP) cells could be responsible for CCK-PZimmunoreactivity. As stressed by Orci et al. (1976a), however, the frequency of glucagon-cells and PP-cells varies in different parts of the rat pancreas. The number of glucagon-cells is conversely related to the number of PP-cells. We could confirm these findings (Fig. 1) concerning the scarcity of glucagon-cells e.g. in the uncinate process of the rat pancreas. In this part of the pancreas CCK-PZ-immunoreactive cells were also encountered relatively seldom. Thus, PP-cells are to be excluded (indeed indirectly, because PP-antisera have not

Immunoreactivity of the Endocrine Pancreas

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been available during our investigations) as the source of CCK-PZ-immunoreactivity in the endocrine pancreas. The final proof for the identity of CCK-PZand glucagon-immunoreactive cells will be given by immunohistochemical investigations of sernithin or ultrathin sections (in prep.). If this identity can be ascertained, the " o n e c e l l - o n e h o r m o n e - t h e o r y " (cf. Solcia et al., 1973) has, besides other consequences, to be considered as obsolete (see also the recent findings of Erlandsen et al., 1976 concerning the presence of somatostatin and gastrin within the same islet cell).

CCK-PZ clnd the Gastro-Entero-Pancreatic Endocrine System The occurrence of CCK-PZ in the pancreas itself, in one of its classical "target organs", has been supposed and even postulated by various authors (Epple and Lewis, 1973; Fujita and Watanabe, 1973; Fujita et al., 1976, ref.). It is interesting to note that CCK-PZ, besides its effects on gallbladder and enzyme output of the pancreas, has a pronounced trophic effect on the exocrine pancreas (Fujita et al., 1976; Johnson, 1976; recent reviews on the actions of CCK-PZ: Jorpes and Mutt, 1973 ; Harvey, 1976 ; Grossman, 1977). These findings together with other pecularities, e.g. the "insulo-acinar portal vessels" (Fujita et al., 1976;see also Ferner, 1952) and the functional subdivision of the exocrine pancreas in peri-insular and tele-insular parts (cf. Malaisse-Lagae et al., 1976, ref.) make it obvious that CCK-PZ, like other islet hormones, may be considered "as local hormones acting upon the exocrine pancreas as their first target" (Fujita et al., 1976). That is to say, the A-cells may influence the peri-insular exocrine tissue by " p a r a c r i n e " release of CCK-PZ as well as by endocrine release of CCK-PZ the tele-insular parts of the exocrine tissue. As to the regulation of the "pancreatic" CCK-PZ-metabolism one might speculate that "enterohormone-releasing factors" (as yet "missing links" in the control system of entero-hormone-metabolism) are released during digestion. Such releasing factors have not to be of peptide structure. Former investigations, for instance, have revealed results which allow the conclusion, that entero-endocrine cells (including A-cells of the pancreatic islets) specifically take up amino acids (Grube, 1976). Thus high levels of different amino acids (resorbed during digestion) may mediate e.g. a CCK-PZ-releasing effect from the gut to the endocrine pancreas (cf. Meyer, 1 9 7 5 ) . - A p a r t from its effects on the exocrine pancreas, CCK-PZ obviously also influences the synthesis rate or release of glucagon and insulin (Raptis et al., 1975; see also Pfeiffer et al., 1973; Unger, 1973). Even though most of the experimental investigations (before 1973/74), concerning the actions of CCK-PZ on the endocrine pancreas, have been performed with poorly purified CCK-PZ-preparations, the presence of CCK-PZ within the islets makes it very likely, that this hormone has also effects on the other islet hormones. As mentioned before, CCK-PZ is not the first gastrointestinal hormone detected in the pancreas. Besides gastrin (cf. Erlandsen et al., 1976), VIP (see Buffa et al., 1977) and secretin (Rufener et al., 1976) also were found in the pancreas. Therefore, one might speculate their most or even all gastrointestinal

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hormones have a counterpart (perhaps molecularly variant) in the pancreas. Moreover, the occurrence of CCK-PZ in the pancreas makes it likely that also the other classical target organ of this hormone, the gallbladder, might contain CCK-PZ-immunoreactive cells. - If further investigations will establish our findings on the occurrence of CCK-PZ and other gastrointestinal hormones in the endocrine pancreas, a new concept on the physiology and interrelationships of gastrointestinal and pancreatic hormones has to be developped. Then the relationships betwen CCK-PZ and pancreatic tumours (Zollinger-Ellison and Verner-Morrison syndrome, cf. Verner and Morrison, 1974; Capella et al., 1977; Dawson, 1976) might also be of special interest. Finally our findings have stressed the close interrelationship between the gastrointestinal endocrine system and the endocrine pancreas on the one hand, and between the endocrine and exocrine part of the pancreas on the other. Acknowledgements. We are indebted to Prof. A. Arimura, New Orleans, USA, for kindly supply with anti-somatostatin. We are grateful to Mrs. G. Krfiutle, Mrs. U. Mann and Miss M. Teufel for their skillful technical assistance.

References Arnold, W., Kalden, J.R., yon Mayersbach, H. : Influence of different histologic preparation methods on preservation of tissue antigens in the immunofluorescence antibody technique. Ann. N.Y. Acad. Sci. 254, 27-34 (1975) Barrington, E.J.W., Dockray, G.J.: Gastrointestinal hormones. J. Endocrinol. 69, 299-325 (1976) Bloom, S.R.: Gastrointestinal hormones. In: Int. Rev. Physiol., Gastrointestinal Physiology II, Vol. 12 (R.K. Crane, ed.), pp. 71-103. Baltimore: University Park Press 1977 Bosman, F.T., Lindeman, J., Kuiper, G., van der Wal, A., Kreunig, J.: The influence of fixation on immunoperoxidase staining of plasmacells in paraffin sections of intestinal biopsy specimens. Histochemistry 53, 57 62 (1977) Braaten, J.T., Greider, M.H., McGuigam J.E., Mintz, D.H.: Gastrin in the perinatal rat pancreas and gastric antrum: immunofluorescence localization of pancreatic gastrin cells and gastrin secretion in monolayer cell cultures. Endocrinology 99, 684 691 (1976) Buffa, R., Solcia, E., Go, V.L.W.: Immunohistochemical identification of the cholecystokinin cell in the intestinal mucosa. Gastroenterology 70, 528-532 (1976) Buffa, R., Capella, C., Solcia, E., Frigerio, B., Said, S.I.: Vasoactive intestinal peptide (VIP) cells in the pancreas and gastrointestinal mucosa. Histochemistry 50, 217-227 (1977) Burger, H., Franchimont, Ch.: Studies on a radioimmunoassay for luteinizing hormone releasing hormone (LH/RH). Horm. Metab. Res., Suppl. 5, 61 65 (1974) Burns, J.: Background staining and sensitivity of the unlabelled antibody-enzyme (PAP) method. Comparison with the peroxidase labelled antibody sandwich method using formalin fixed paraffin embedded material. Histochemistry 43, 291 294 (1975) Capella, C., Solcia, E., Frigerio, B., Buffa, R., Usellini, L., Fontana, P.: The endocrine cells of the pancreas and related tumours. Virchows Arch. (Pathol. Anat.) 373, 327-352 (1977) Coons, A.H., Leduc, E.H., Conolly, J.: Studies on antibody production. I. A method for the histochemical demonstration of specific antibody and its application to a study of the hyperimmune rabbit. J. exp. Med. 102, 49 60 (1955) Dawson, I.M.P.: The endocrine cells of the gastrointestinal tract and the neoplasms which arise from them. Curr. Top. Pathol. 63, 221-258 (1976) Dockray, G.J., Best, L., Taylor, I.L.: Immunochemical characterization of gastrin in pancreatic islets of normal and genetically obese mice. J. Endocrinol. 72, 143 151 (1977) Dubois, P.M., Paulin, C., Chayvialle, J.A. : Identification of gastrin-secreting cells and cholecystoki-

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nin-secreting cells in the gastrointestinal tract of the human fetus and adult man. Cell Tissue Res. 17B, 351-356 (1976) Ekins, R.P.: Basic principles and theory (radioimmunoassay and saturation analysis). Brit. reed. Bull. 30, 3-11 (1974) Epple, A., Lewis, T.L. : Comparative histophysiology of the pancreatic islets. Am. Zool. 13, 567 590 (1973) Erlandsen, S.L., Hegre, O.D., Parsons, J.A., McEvoy, R.C., Elde, R.P.: Pancreatic islet cell hormones. Distribution of ceil types in the islet and evidence for the presence of somatostatin and gastrin within the D-ceIi. J. Histochem. Cytochem. 24, 883-897 (1976) Falck, B., Hillarp, N.A., Thieme, G., Torp, A.: Fluorescence of catecholamines and related compounds condensed with formaldehyde. J. Histochem. Cytochem 10, 348-354 (1962) Ferner, H. : Das Inselsystem des Pankreas, pp. 96-103. Stuttgart: Georg Thieme 1952 Forssmann, W.G., Yanaihara, N., Hehnstaedter, V., Orube, D. :Differential demonstration of the motilin-cell and the enterochromaffin cell. Scand. J. Gastroent. 11, Suppl. 39, 43-45 (1976) Fujita, T. (ed.): Gastro-entero-pancreatic endocrine system. Tokyo: Igaku Shoin 1973 Fujita, T., Watanabe, Y.: The effects of islet hormones upon the exocrine pancreas. In: Gastroentero-pancreatic endocrine system (T. Fujita, ed.), pp. 164-t73. Tokyo: Igaku Shoin 1973 Fujita, T. (ed.): Endocrine gut and pancreas. Amsterdam-New York: Elsevier 1976 Fujita, T., Yanatori, Y., Murakami, T.: Insulo-acinar axis, its vascular basis and its functional and morphological changes caused by CCK-PZ and caerulein. In: Endocrine gut and pancreas (T. Fujita, ed.), pp. 347-357. Amsterdam-New York: Elsevier I976 Graham, B.C., Karnovsky, M.J. : The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructnral cytochemistry by a new technique. J. Histochem. Cytochem. 14, 281-302 (1966) Grossman, M.I. : Physiological effects of gastrointestinal hormones. Fed. Proc. 36, 1930-1932 (1977) Grube, D.: Die endokrinen Zellen des Magendarmepithels und der Stoffwechsel der biogenen Amine im Magendarmtrakt. Progr. Histochem. Cytochem. 8/3, 1 128 (1976) Grube, D., Helmstaedter, V., Feurle, G., Yanaihara, N., Forssmann. W.G. : Die entero-endokrinen ZelIen des Spitzh6rnchens (Tupaia be/angeri). Verh. Anat. Ges. 71, 1137-1143 (I977) Harvey, R.F. : Radioimmunoassay of CCK-PZ. Gut 15, 690-699 (i974) Harvey, R.F.: Physiology and pathophysiology of cholecystokinin-pancreozymin. J. Endocrinol. 70, 5P-6P (1976) Heding, L.G., Rasmussen, S.M. : Determination of pancreatic and gut glucagon-like immunoreactivity (GLI) in normal and diabetic subjects. Diabetologia K 408-411 (1972) Herbert, V., Lau, K.S, Gottlieb~ C.W., Bleicher, S.J.: Coated charcoal immunoassay of insulin. J. clin. Endocrinol. Metab. 25. 1375-1384 (1965) H6kfelt, %, Efendic, S., Hellerstr6m~ C.~ Johansson, O., Luft, R., Arimura, A. : Cellular localization of somatostatin in endocrine-like cells and neurons of the rat with special references to the AI cells of the pancreatic islets and to the hypothalamus. Acta endocrinol. (Kbh), Suppl. 200, 5-41 (1975) Ito, S., Kobayashi, S. : Immunohistochemical demonstration of glucagon- and GLI-containing cells in the canine gut and pancreas. Arch. histol, jap. 3K 193-202 (1976) Johnson, L.R.: The trophic action of gastrointestinal hormones. Gastroenterology 70, 278-288 (1976) Jorpes, J.E., Mutt, V. : Secretin and cholecystokinin (CCK). In : Secretin, cholecystokinin, pancreozyrain and gastrin. Handbook of experimental Pharmacology, Vol. 34 (J.E. Jorpes, V. Mutt, ed.), pp. 1-179. Berlin-Heidelberg-New York: Springer 1973 Lange, R.H.: Histochemistry of the islets of Langerhans. In: Handbuch der Histochemie, Vol. VIII/1 (W. Graumann, K. Neumann, ed.), pp. 1-141. Stuttgart: G. Fischer 1973 Larsson, L.I.. Holst, J., Hflkanson, R , Sundler, F. : Distribution and properties of glucagon immunoreactivity in the'digestive tract of various mammals: an immunohistochemical and immunochemical study. Histochemistry 44, 281-290 (1975) Lotstra, F., van der Loo, W., Gepts, W. : Are gastrin-cells present in mammalian pancreatic islets ? Diabetologia 10, 291-302 (1974) Malaisse-Lagae, F., Ravazzola, M., Robberecht, P., Vandermeers, A., Malaisse, W.J., Orci, L.: Hydrolase content in peri-insular and teD-insular exocrine pancreas. In: Endocrine gut and pancreas (T. Fujita, ed.), pp. 313 320, Amsterdam-New York: Elsevier 1976

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McGuigan, J.E.: Pancreatic and extra-pancreatic gastrin. In: Handbook of Physiology, Sect. 7 (D.F. Steiner and N. Freinkel, ed.), pp. 279-287. Washington: American Physiological Society 1972 Meyer, J.H. : Release of secretin and cholecystokinin. In: Gastrointestinal hormones (J.C. Thompson, ed.), pp. 475-489. Austin: University of Texas Press 1975 Moody, A.J., Jacobson, H., Sundby, F.: Gastric glucagon and gut glucagon-like immunoreactants. In: Proc. int. Symp. " G u t hormones", Lausanne 1977. Edinburgh: Churchill Livingstone (in press) Mutt, V.: Further investigations on intestinal hormonal polypeptides. Clin. Endocrinoh (Oxford) 5, Suppl. 175s 183s (1976) Nakane, P.K.: Simultaneous localization of multiple tissue antigens using the peroxidaseqabeled antibody method: a study on pituitary glands of the rat. J. Histochem. Cytochem. 16, 557-560 (1968) Orci, L., Baetens, D., Ravazzola, M., Stefan, Y., Malaisse-Lagae, F.: Pancreatic polypeptide and glucagon: non-random distribution in pancreatic islets. Life Sci. 19, 1811-1816 (1976a) Orci, L., Baetens, D., Rufener, C., Amherdt, M., Ravazzola, M., Studer, P., Malaisse-Lagae, F., Unger, R.H.: Hypertrophy and hyperplasia of somatostatin-conataining D-cells in diabetes. Proc. nat. Acad. Sci. 73, 1338-1342 (1976b) Parsons, J.A., Erlandsen, S.L., Hegre, O.D., McEvoy, R.C., Elde, R.P.: Central and peripheral localization of somatostatin. Immunoenzyme immunocytochemical studies. J. Histochem. Cytochem. 24, 872 882 (1976) Pearse, A.G.E., Polak, J.M., Adams, C., Kendall, P.A.: Diethylpyrocarbonate, a vapour-phase fixative for immunofluorescence studies on polypeptide hormones. Histochem. J. 6, 347-352 (1974) Pearse, A.G.E., Polak, J.M., Bloom, S.R.: The newer gut hormones. Cellular sources, physiology, pathology, and clinical aspects. Gastroenterology 72~ 746-761 (1977) Petrusz, P., Sar, M., Ordronneau, P., Dimeo, P.: Specificity in immunocytochemical staining. J. Histochem. Cytochem. 24, 1110-1115 (1976) Petrnsz, P., Sar, M., Ordronneau, P., Dimeo, P. : Reply to the letter of Swaab et al. : "Can specificity ever be proved in immunocytochemical stainiug?"J. Histochem. Cytochem. 25, 390-391 (1977) Pfeiffer, E.F., Raptis, S., Ful3g~nger, R.: Gastrointestinal hormones and islet function. In: Handbook of experimental Pharmacology, Vol. 34 (J.E. Jorpes and V. Mutt, ed.), pp. 259-310. Heidelberg-Berlin-New York: Springer 1973 Piris, J., Whitehead, R. : An immunoperoxidase technique for the identification of gastrin-producing ceils, J. clin. Pathot. 27,798 799 (1974) Pointner, H. : Extraktion und Charakterisierung eines gastro-sekretagogen Peptids aus dem Pankreas. Wien. klin. Wschr. 87, 406-418 (1975) Polak, J.M., Bloom, S.R., Rayford, P.L, Pearse, A.G.E., Buchan, A.M.J., Thompson, J.C. : Identification of cholecystokinin-secreting cells. Lancet 1975/I1, 1016-1018 Raptis, S., Dollinger, H.C., Schlegel, W., Nadjafi~ A.S. : Portal and peripheral blood concentrations of insulin and exocrine pancreatic secretion in response to pure (99%) cholecystokinin-pancreozyrain in man. Diabetologia 11, 371 (1975) Rayford, P.L., Miller, T.A., Thompson, J.C.: Secretin, cholecystokinin and newer gastrointestinal hormones. New England J. Med. 294, 1093-1101 (1976a) Rayford, P.L., Miller, T.A., Thompson, J.C.: Secretin, cholecystokinin and newer gastrointestinal hormones. New England J. Med. 294, 1157 1164 (1976b) Reading, M. : A digestion technique for the reduction of background staining in the immunoperoxidase method. J. clin. Pathol. 30, 88-90 (1977) Rufener, C., Amherdt, M., Baetens, D., Yanaihara, N., Orci, L.: Immunofluorescent localization of secretin in pancreatic monolayer culture. Histochemistry 47, 171-173 (1976) Schlegel, W., Raptis, S. : A reliable method for generating antibodies against pancreozymin, secretin and gastrin. Clin. chim. acta 73, 439-444 (1976) Schlegel, W., Raptis, S., Grube, D., Pfeiffer, E.F.: Estimation of cholecystokinin-pancreozymin (CCK) in human plasma and tissue by a specific radioimmunoassay and the immunohistochemical identification of pancreozymin producing cells in the duodenum of humans. Clin. china. acta 80, 305-316 (1977) Solcia, E,, Pearse, A.G.E., Grube, D., Kobayashi, S., Bussolati, G., Creutzfeldt, W., Gepts, W.:

Immunoreactivity of the Endocrine Pancreas

35

Revised Wiesbaden classification of gut endocrine cells. Rendic. Gastroenterology 5, 13-16 (1973) Sternberger, L.A. : Immunocytochemistry. Englewood Cliffs, N.J. : Prentice-Hall 1974 Sundler, F., H~.kanson, R., Lundquist, I., Larsson, L.I. : Effect of Alloxan on rat pancreatic polypeptide (PP) cells. Cell Tissue Res. 178, 307-312 (1977) Swaab, D.F., Pool, C.W., Van Leeuwen, F.W,: Can specificity ever be proved in immunocytochemical staining? J. Histochem. Cytochem. 25, 388-391 (1977) Taylor, C.R.: The nature of Reed-Sternberg cells and other malignant "reticulum" cells. Lancet 1974/II, 802-806 Unger, R.H.: The gastrointestinal hormones as modifiers of islet cell hormone secretion. In: Frontiers in gastrointestinal hormone research (S. Anderson, ed.), pp. 259-269. Stockholm: Almquist & Wiksell 1973 Unger, R.H., Orci, L.: Possible roles of the pancreatic D-cell in the normal and diabetic states, Diabetes 26, 241-244 (1977) Unger, R.H., Srikant, C.B., Baetens, D.: Gastrointestinal glucagon and GLL In: Endocrine gut and pancreas (T. Fujita, ed.), pp. 219-224. Amsterdam-New York: Elsevier 1976 Verner, V., Morrison, A.B.: Endocrine pancreatic islet disease with diarrhea. Arch. int. Med. 133, 492-500 (1974) Yalow, R.S., Straus, E.: Heterogeneity of gastrointestinal hormones. In: I.N.S.E.R.M. Symposium No. 3 (S. Bonfils, P. Fromageot, G. Rosselin, ed.), pp. 79-93. Amsterdam-New York: Elsevier 1977

Received January 2, 1978

Note added in proof."

Using semithin serial sections ofepo• rat pancreatic tissue for immunocytochemical investigations it could be confirmed that CCK-PZ-immunoreactivity is confined to the A-(glucagon-) cells.