Effect of bombesin-stimulated gastrin on gastric acid secretion in man

The effect of bombesin on gastrin release and gastric acid secretion was investigated in 10 healthy volunteers. Bombesin (0.6 μg · Kg^?1 · hr^?1) produced a significantly higher $\text{(}\text{p}\text{<}\text{0.001)}$ increase in plasma gastrin levels (86.7 11.1 pmo/1 than after a protein meal (39.6 ± 5.6 pmol1/1). The gastric acid secretory response to bombesin (12.1 ± 2.9 mEq · hr^?1) was however significantly lower $\text{(}\text{p}\text{<}\text{0.005)}$ than the maximal response produced by pentagostrin (20.9 ± 3.5 mEq · hr^?1) at the dose of 6 μg · Kg^?1. Atropine did not modify gastrin release induced by bombesin but significantly reduced gastric acid secretion $\text{(}\text{p}\text{<}\text{0.01)}$. From the data presented it may be hypothesized that less biologically active forms of gastrin and/or other peptides inhibiting the gastrin effect upon gastric acid secretion may be released by bombesin.     

Predicting Damage of Meloidogyne incognita on Watermelon

Quantitative growth response of watermelon (Citrullus lanatus) sensitive to Meloidogyne incognita is poorly understood. Determination of soil population densities of second-stage juveniles (J2) of M. incognita with Baermann funnel extraction often is inaccurate at low soil temperatures. In greenhouse experiments, three sandy soils were inoculated with dilution series of population densities of eggs or J2 of M. incognita and planted in small containers to watermelon ‘Royal Sweet’ or subjected to Baermann funnel extraction. After five weeks of incubation in the greenhouse bioassay plants in egg-inoculated soils, gall numbers on watermelon roots related more closely to inoculated population densities than J2 counts after Baermann funnel extraction. In April 2004, perpendicularly-inserted tubes (45-cm diameter, 55-cm deep) served as microplots where two methyl bromide-fumigated sandy soils were inoculated with egg suspensions of M. incognita at 0, 100, 1,000 or 10,000 eggs/100 cm³ of soil in 15-cm depth. At transplanting of 4-week old watermelon seedlings, soils were sampled for the bioassay or for extraction of J2 by Baermann funnel. In the Seinhorst function of harvested biomass in relation to nematode numbers, decline of biomass with increasing population densities of M. incognita was accurately modeled by the inoculated eggs (R² = 0.93) and by the counts of galls on the bioassay roots (R² = 0.98); but poorly by J2 counts (R² = 0.68). Threshold levels of watermelon top dry weight to M. incognita were 122 eggs/100 cm³ soil, 1.6 galls on bioassay roots, or 3.6 J2/100 cm³ of soil. Using the bioassay in early spring for predicting risk of nematode damage appeared useful in integrated pest management systems of watermelon.     

Growth of mouse hepatocytes is stimulated by gastrin

Hepatocyte growth is regulated by various growth factors, including epidermal growth factor (EGF) and insulin. Recently, several additional peptide hormones have been shown to stimulate growth of hepatocyte only in the presence of EGF or insulin and are thus termed secondary mitogens. Gastrin regulates growth of normal and neoplastic gastrointestinal tissues, but the effect on growth of hepatocyte is unknown. We examined the effect of gastrin on growth of a normal mouse hepatocyte (NMH) line established in our laboratory. Effect of gastrin-17 (G-17) (10^?8 to 10^?6 M) on growth of NMH cells was examined in either the presence or absence of EGF in the culture medium. Growth of NMH cells was evaluated by incorporation of either bromodeoxyuridine (BrdU) or ³H-thymidine and by counting cells. Presence of a cell-surface receptor for G-17 was determined by Scatchard analysis using ¹²⁵I-G-17. In the presence of EGF, gastrin stimulated growth of NMH cells; in the absence of EGF, gastrin did not affect growth. The stimulatory effect of gastrin on NMH cells was blocked by JMV 320, a CCK-B type receptor antagonist. NMH cells possess a single, high affinity binding site for gastrin (K_d = 1.2 nM); EGF increased the gastrin binding capacity compared to non-treated cells (3.5 ± 0.4 vs. 2.2 ± 0.6 fmol/10⁶ cells). G-17 stimulated growth of NMH cells through a single high affinity receptor for G-17 which pharmcologically appears to be the CCK-B type only in the presence of EGF and thus can be considered a secondary mitogen. © 1995 Wiley-Liss, Inc.     

Interaction of calcium ions with gastrin fragments of increasing chain length

The interaction of a series of biologically active gastrin fragments with calcium ions has been investigated by CD in trifluoroethanol. It was found that the gastrin octapeptide pGlu¹⁰,Nle¹⁵-HG[10–17] binds one calcium ion per molecule. The hypothesis is made that the binding involves the C-terminal, biologically important tetrapeptide. When the chain is elongated to the gastrin nonamer pGlu⁹,Nle¹⁵-HG[9–17], a second binding site is available, which is most likely situated at the N-terminal part of the molecule. Further elongation of the peptide chain up to the dodecapeptide pGlu⁶,Nle¹⁵-HG[6–17] does not provide any additional binding site. Saturation of the two sites in the shorter peptides produces different changes in the chiroptical properties in the near- and far-uv. As the chain is elongated, this difference tends to disappear. This result is consistent with an increased conformational order of the longer peptides. In the shorter fragments, the strength of this second binding is appreciably lower than that of the first, while in the longer peptides, the strength of the two bindings is comparable. On the assumption that the variation of the CD properties is proportional to the extent of binding, the constant for the binding of the second ion was determined to be of the order of 5 × 10⁵ L/mol for the nonapeptide.     

Biologically active gastrin/CCK-related peptides in the stomach of a reptile, Crocodylus niloticus; Identified and characterized by immunochemical methods

We have used immunochemical, chromatographic, and bioassay techniques to characterize peptides related to gastrin and CCK, from the stomach of the reptile Crocodylus niloticus. By immunocytochemistry gastrin/CCK-like peptides were localized in specific mucosal cells of the pylorus and in the duodenum. Boiling water extracts of pyloric antrum cross reacted with four antisera specific for the C-terminal region of gastrin or CCK, but estimates of concentration varied between antisera. Antisera specific for the N-terminus of heptadecapeptide gastrin (G17), intact G17, or the amphibian CCK-like peptide caerulein did not cross react with the crocodile extracts. Gel filtration of the extracts on Sephadex G50 resolved one major peak eluting significantly before G17 or CCK8, suggesting larger molecular size, whereas ion exchange on DE52 cellulose resolved two major immunoreactive peaks, both eluting before G17, indicating that they are less acidic. The more acidic of the two peptides stimulated gastric acid secretion in the rat, but had no CCK-like actions on the rat pancreas. Thus crocodile antrum contains gastrin-like peptides, which are however clearly distinguishable from any of the known mammalian forms of gastrin and CCK.     

A new model for sensitive detection of zoonotic prions by PrP transgenic Drosophila

Prions are transmissible protein pathogens most reliably detected by a bioassay in a suitable host, typically mice. However, the mouse bioassay is slow and cumbersome, and relatively insensitive to low titers of prion infectivity. Prions can be detected biochemically in vitro by the protein misfolding cyclic amplification (PMCA) technique, which amplifies disease-associated prion protein but does not detect bona fide prion infectivity. Here, we demonstrate that Drosophila transgenic for bovine prion protein (PrP) expression can serve as a model system for the detection of bovine prions significantly more efficiently than either the mouse prion bioassay or PMCA. Strikingly, bovine PrP transgenic Drosophila could detect bovine prion infectivity in the region of a 10⁻¹² dilution of classical bovine spongiform encephalopathy (BSE) inoculum, which is 10⁶-fold more sensitive than that achieved by the bovine PrP mouse bioassay. A similar level of sensitivity was observed in the detection of H-type and L-type atypical BSE and sheep-passaged BSE by bovine PrP transgenic Drosophila. Bioassays of bovine prions in Drosophila were performed within 7 weeks, whereas the mouse prion bioassay required at least a year to assess the same inoculum. In addition, bovine PrP transgenic Drosophila could detect classical BSE at a level 10⁵-fold lower than that achieved by PMCA. These data show that PrP transgenic Drosophila represent a new tractable prion bioassay for the efficient and sensitive detection of mammalian prions, including those of known zoonotic potential.     

Murine CD4 T cells produce a new form of TGF-β as measured by a newly developed TGF-β bioassay

Background

It is generally assumed that T cells do not produce active TGF-β since active TGF-β as measured in supernatants by ELISA without acidification is usually not detectable. However, it is possible that active TGF-β from T cells may take a special form which is not detectable by ELISA.

Methodology/Principal Findings

We constructed a TGF-β bioassay which can detect both soluble and membrane-bound forms of TGF-β from T cells. For this bioassay, 293T cells were transduced with (caga)₁₂ Smad binding element-luciferase along with CD32 (Fc receptor) and CD86. The resulting cells act as artificial antigen presenting cells in the presence of anti-CD3 and produce luciferase in response to biologically active TGF-β. We co-cultured pre-activated murine CD4⁺CD25⁻ T cells or CD4⁺CD25⁺ T cells with the 293T-caga-Luc-CD32-CD86 reporter cells in the presence of anti-CD3 and IL-2. CD4⁺CD25⁻ T cells induced higher luciferase in the reporter cells than CD4⁺CD25⁺ T cells. This T cell-produced TGF-β is in a soluble form since T cell culture supernatants contained the TGF-β activity. The TGF-β activity was neutralized with an anti-mouse LAP mAb or an anti-latent TGF-β/pro-TGF-β mAb, but not with anti-active TGF-β Abs. An anti-mouse LAP mAb removed virtually all acid activatable latent TGF-β from the T cell culture supernatant, but not the ability to induce TGF-β signaling in the reporter cells. The induction of TGF-β signaling by T cell culture supernatants was cell type-specific.

Conclusions/Significance

A newly developed 293T-caga-Luc-CD32-CD86 reporter cell bioassay demonstrated that murine CD4 T cells produce an unconventional form of TGF-β which can induce TGF-β signaling. This new form of TGF-β contains LAP as a component. Our finding of a new form of T cell-produced TGF-β and the newly developed TGF-β bioassay system will provide a new avenue to investigate T cell function of the immune system.     

Conformational aspects of gastrin-related peptides: A circular dichroism study

The conformational properties of a series of gastrin-related peptides in aqueous solution and in 2,2,2-trifluoroethanol (TFE) have been investigated by CD measurements. In aqueous solution the peptides Leu³²-HG-34 (human big gastrin), Nle¹⁵-HG-17 (human little gastrin), and Nle¹¹-HG-13 assume a random-coil structure in the pH range 3–7. In TFE the three hormones fold into partially ordered structures, consisting of mixtures of α-helix, β-form and random coil. Comparison with the CD properties of the shorter gastrin peptides HG-4 (tetragastrin), N^α-Boc-HG-5 (pentagastrin), and HG-7 (heptagastrin) indicates that the biologically important C-terminal sequence Trp-Met-Asp-Phe-NH₂ in TFE does not maintain the same geometry upon elongation of the chain at the N-terminus from 4 to 34 residues. Thus, the various conformations in solution of the gastrin peptides examined do not provide a structural explanation for their very similar biological activity. Therefore, we hypothesize that the C-terminal tetrapeptide amide folds into an “active” structure only upon interaction with the receptor.     

High affinity binding sites for somatostatin to rat pituitary

Binding sites for somatostatin (SS) are described in rat pituitary membranes using either [¹²⁵I-Tyr¹¹]-SS-14 or [Leu⁸, D-Trp²², ¹²⁵I-Tyr²⁵]-SS-28 as radioligands; in each case saturable and high affinity binding sites with K_D's for SS of 1.09 and 0.95 nM respectively have been characterized. The binding capacity is 100 f mols/mg protein. The potencies of various SS analogs measured in the radioreceptor assay are in agreement with the potencies in a bioassay measuring inhibition of growth hormone release; in particular, SS-28 is slightly less potent than SS-14. A comparison of these data with those describing SS binding in brain and pancreas suggests that some pharmacological differences may exist between pituitary, brain and pancreas binding sites for SS.     

Vasoactive intestinal peptide (VIP) cells in the pancreas and gastro-intestinal mucosa

Summary Using antibodies against pure porcine VIP in immunoperoxidase and immunofluorescence tests, VIP-immunoreactive cells have been detected in the pancreas—especially in the islets—and gastrointestinal mucosa of the dog, guinea-pig and man. VIP immunoreactive cells were widely distributed in these tissues, never being numerous at any site. Some parallelism has been noted between such cells and ultrastructurally identified D₁ cells of the pancreas and gastrointestinal mucosa. The presence of VIP cells in normal pancreas may help explain the occurrence of pancreatic endocrine tumors producing VIP.     

Assignment of <Emphasis Type="Italic">Lemna gibba</Emphasis> L. (duckweed) bioassay for <Emphasis Type="Italic">in situ</Emphasis> ecotoxicity assessment

To narrow the differences between the results obtained from radionuclides and heavy metal ecotoxicity investigations in the laboratory and in the abandoned uranium mines, a few standardised plant bioassay procedures were selected from the literature for testing with Lemna gibba L. The bioassay procedures were tested in situ and ex situ. The laboratory culturing was performed in batch and semicontinuous modes. The results revealed that most of the standardised plant bioassay procedures require modification for the L. gibba bioassay to predict the actual effects under field conditions. L. gibba performed relatively better in the field than laboratory batch cultures despite that the batch cultures had many-fold higher nutrient concentrations than in the field. For instance, the phosphorus concentration of the mine tailing water was 0.13 ± 0.09 μg l⁻¹ in the field, while the literature range for phosphorus in the laboratory culture media is 13.6–40 mg l⁻¹. L. gibba growth in the laboratory batch culture was influenced by speciation changes due to consumption of nutrients, CO₂ and O₂ phase exchanges, and excretion of organic substances by the test plants. Semicontinuous culture modes performed significantly better than batch cultivation even after 10× dilution of the nutrient solution. The growth behaviour revealed that L. gibba exhibited intrapopulation and probiotic interaction for best performance. Growth performance of L. gibba was influenced by the anions that balanced essential cations despite equal cation concentration in the culture media; e.g., the best growth was observed in culture media that had more SO₄²⁻ than Cl⁻. Water samples from the field had higher SO₄²⁻ concentrations than Cl⁻. The test vessel material, sterilisation and axenic culturing procedures also influenced the sensitivity of the bioassay. These, for instance, and a few others are neither described nor reported in most standard Lemna tests or the literature. Thus, this work presents results of a series of tests conducted on the selected methods. Common and possible errors and corrective measures in assigning L. gibba bioassay from laboratory population levels to field community levels are discussed.     

Chemical characterisation,antioxidant and antimicrobial screening for the revaluation of wine supply chain by-products oriented to circular economy

Abstract

Aim of the project was the bioassay guided optimisation of extraction methods applied to wine chain by-product to obtain extracts, fractions and biologically active biomolecules with a possible use in the nutraceutical and cosmeceutical industry. Exhausted red and white grape marc were extracted using water:ethanol 50:50 with ultrasound assisted extraction and Naviglio® technology; and also with supercritical fluid extraction (SFE) and steam-distillation obtaining different phytocomplexes. Each extract was characterised by different molecular category: exhausted red grape marc (VCR) by anthocyanins, exhausted white grape marc (VCB) by flavonoids, and grapeseed (VIN) by proanthocyanins. SFE and steam-distillation, instead, highlighted the presence of fatty acids and their ester in every matrix, but terpenoids were not revealed at level major or equal than 0.1%, except for manoyl oxide in VCR (2.89%). VIN was the most abundant matrix in polyphenols (506.24?±?55.91?mg gallic acid/g dried extract), and it showed the highest antioxidant activity (IC₅₀ of 4.30?μg/mL). Regarding the antimicrobial activity, the hydroalcoholic extracts from VCR, VCB and VIN were tested but no noteworthy activities have been recorded.     

High potency of bombesin for stimulation of human gastrin release and gastric acid secretion

Dose-response studies were performed in 6 human volunteer subjects to determine the threshold and optimal doses of intravenous bombesin for stimulation of gastric acid secretion and gastrin release. A significant stimulation of both acid and gastrin was obtained with a very low dose, 3 pmol · kg^?1 · h^?1. Peak stimulation of acid secretion (67% of pentagastrin PAO) was obtained at 12.5 pmol · kg^?1 · h^?1. Serum gastrin response to this dose of bombesinn was similar to that obtained after a high protein meal. Higher doses of bombesin caused further increases in serum gastrin but not in acid secretion. Since very low doses of bombesin, too small to produce detectable increases in immunoreactive serum bombesim, caused parallel increases in gastrin and acid secretion, it is possible that the bombesin-like peptides present in human gastrointestinal tissues contribute to regulation of human gastric secretion.     

Serpina3c protects against high-fat diet-induced pancreatic dysfunction through the JNK-related pathway

BackgroundSerpina3 is a member of the serine protease inhibitor family and is involved in the inflammatory response. In this study, we investigated the effect of Serpina3c on pancreatic function in hypercholesterolemic mice.MethodsTo investigate the role of Serpina3c in hyperlipidaemia, Serpina3c knockout mice were bred with Apoe-knockout mice (on a C57BL/6 background) to generate heterozygous Serpina3c-Apoe double knockout (Serpina3c^+/−/Apoe^+/−) mice and were then bred to obtain homozygotes. C57BL/6, Serpina3c^−/−, Apoe^−/−, and Apoe^−/-Serpina3c^−/− mice were fed normal chow, and Apoe^−/− and Apoe^−/-Serpina3c^−/− mice were fed a high-fat diet (HFD). After feeding for 3 months, the mice were monitored for body weight, blood glucose, glucose tolerance, and insulin tolerance test (ITT). ELISA and immunohistochemistry were used to detect insulin levels and glucagon expression. Immunohistochemical staining for macrophages in the pancreas was also performed. Western blot analysis was performed on pancreatic tissues to detect the protein levels of insulin-associated molecules, the metalloproteinase MMP2, the tissue inhibitor TIMP2 and components of the JNK-related pathway.ResultsBlood glucose levels, glucose tolerance, and ITT were not significantly different among the groups. Serpina3c knockout resulted in blood lipid abnormalities in mice under HFD conditions. Insulin secretion was decreased in Apoe^−/-Serpina3c^−/− mice compared with Apoe^−/− mice under normal chow conditions. In addition, Apoe^−/-Serpina3c^−/− mice exhibited increased insulin and glucagon secretion and expression after three months of HFD feeding, but insulin secretion was decreased in Apoe^−/-Serpina3c^−/− mice compared with Apoe^−/− mice after the fifth month of HFD feeding. Serpina3c knockout increased MMP2 protein levels, whereas TIMP2 levels in the pancreas were decreased. Furthermore, Serpina3c knockout significantly upregulated the number of macrophages in the pancreas under HFD conditions. The JNK/AKT/FOXO1/PDX-1 axis was found to be involved in Serpina3c-regulated insulin secretion.ConclusionThese novel findings show that Serpina3c could play a protective role in insulin secretion partly through the JNK-related pathway under HFD conditions.     

Expression of alien histocompatibility antigens on SJL/J tumors detected by cell-mediated and serological analyses

Summary Reticulum cell sarcoma (RCS) cells of SJL/J (H-2^s) mice have been shown to express antigens that are cross-reactive with allogeneic cells of the H-2^d and H-2^b haplotypes by cell-mediated cytotoxicity, antibody-mediated cytotoxicity, immunofluorescence, and quantitative absorption assays. These alien antigens have been detected on both spontaneous and in vivo- and in vitro-passaged RCS cells to varying degrees.The in vitro cell lines were able to stimulate a syngeneic cytotoxic T cell response detected in a 4-h ⁵¹Cr release assay. The cytotoxic cells reacted with in vitro RCS tumor targets but not with in vivo or spontaneous RCS tumors. Furthermore, the cytotoxic cells lysed H-2^d and to a lesser extent H-2^b target cells, but not H-2^k, H-2^p, or H-2^r cells. The cross-reactivity was also observed with SJL/J anti-BALB/c cytotoxic cells, which can lyse in vitro RCS targets effectively. The in vivo tumors were not stimulatory in cytotoxic responses and did not serve as targets.H-2^d specificities were also detected in cultured RCS tumor cells by cytotoxic antibody. Both allogeneic SJL/J anti-BALB/c, C57B1/6 anti-BALB/c sera reacted with RCS tumor cells and not normal SJL/J cells. Furthermore, monospecific D^d sera were also cytotoxic against RCS lines. The cytotoxic activity could be absorbed by BALB/c cells and RCS cells but not with normal SJL/J cells. The H-2^d specificities were also detected on the in vivo lines by indirect immunofluorescence. The majority (60%) of spontaneously arising tumors expressed either H-2^d or H-2^b allospecificities in the immunofluorescence assays. Although these antigens may not be inappropriate for the SJL/J strain, their differential expression on tumor cells may be significant in the etiology of the tumor.     

Biological activity of des-(Asp1, Arg2, Val3)-angiotensin II in man

When des-(Asp¹, Arg², Val³)-angiotensin II was infused iv at rates of 308–5,550 pmol/kg·min for 10–120 min into 5 normal men and 2 patients with Bartter's syndrome, no significant change was observed in blood pressure (BP), plasma renin activity (PRA) or plasma aldosterone (PA), and the lowest dose did not inhibit a captopril-induced increase in PRA in the normal men, although des-(Asp¹, Arg²)-angiotensin II was reported in the same 5 normal men to cause a decrease in PRA and an increase in PA in this dose range and a rise in BP at 2,220 and 5,550 pmol/kg·min. However, an iv infusion of the pentapeptide at 9,000 pmol/kg·min for 15 min significantly raised BP in the 5 normal men but not in patients with Bartter's syndrome. BP returned to the pretreatment level 60 min after the cessation of the infusion, although the duration of the pressor actions of angiotensin II, angiotensin III and des-(Asp¹, Arg²)-angiotensin II were reported to be within 5 min in man. At the same dose level none of the 7 examined subjects showed any significant change in PRA or PA. Des-(Asp¹, Arg², Val³, Tyr⁴)-angiotensin II was infused iv at a rate of 41,480 pmol/kg·min into one of the normal men, but it caused no significant change in BP, PRA or PA. These results suggest that the pentapeptide and probably the tetrapeptide do not possess renin-suppressing and steroidogenic actions in man but the pentapeptide does elecit a minimal pressor action with a prolonged duration.     

The G-cells in the dog: a light and electron microscope immunocytochemical study

Summary An immunohistochemical study has been performed to analyse the distribution of gastrin cells in the gastrointestinal tract of the dog. This study revealed that G-cells immunoreactive for gastrin were almost exclusively present in the pyloric antral mucosa, mainly in the middle third of the pyloric mucosa. The calculated number of G-cells per surface unit area was 8.5×10³–1.2×10⁴ cells cm^–2. Some gastrin-immunopositive cells were found in the first 10 mm of the proximal duodenum, mainly in the villous region. The fundic area of the dog stomach, the oesophagus, small intestine, caecum, colon, rectum, salivary glands, liver and pancreas were all immunonegative for gastrin. At the ultrastructural level, three different types of granules (150–400 nm) were evident in G-cells: electron-dense, electron-lucent and intermediate forms. Most of them were located in the subnuclear region of the cell. The effect of fixation of the antral mucosa at different pH levels was studied. In samples fixed with acid solutions, most of the G-cell granules were of the electron-dense type and were stronly immunopositive for gastrin. Fixation of samples at a basic pH resulted in most of the gastrin granules losing their contents into the cytoplasm, and the positive reaction to gastrin was then located in the cytoplasm and at the periphery of the electron-lucent granules.     

A specific gastrin receptor site in the rat stomach

A specific receptor for gastrin I has been demonstrated in the rat stomach fundus.Specific binding of ¹²⁵I-labelled gastrin I was localised to particles sedimenting between 250–20 000 × g. Saturation of binding sites occurred with a gastrin concentration of 10^?11 M in an assay system containing 0.6–1.7 mg/ml of homogenate protein. Gastrin binding was shown to be reversible, temperature- and pH-dependent, and susceptible to tryptic digestion. Electron microscopic and enzymatic studies showed the binding fraction to contain predominantly mitochondria. Preincubation of the homogenate with 10^?8 M cholecystokinin or secretin inhibited gastrin binding to a greater extent than an equimolar concentration of pentagastrin. Cimetidine, a histamine receptor antagonist, did not affect binding of gastrin to the receptor.     

Immunoreactivity of the endocrine pancreas. Evidence for the presence of cholecystokinin-pancreozymin within the A-cell

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 demon-strated. 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. From 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 adsorbed 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 proof of immunohistochemical findings. — The 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. From the enterohormones gastrin, secretin and CCK-PZ only CCK-PZ (or a CCK-PZ-like peptide) was found to occur in the human and rat pancreas. The presence of CCK-PZ in the pancreas was also confirmed by radioimmunological estimations of CCK-PZ 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 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 one cell — one hormone theory in question. — Gastrin- and 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 radioimmunoassay. — The occurrence of CCK-PZ, a classical enterohormone, in its target organ 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.Supported by a grant of the German Research Foundation, SFB 87, Ulm, FRG