Distribution of gastrin and CCK cells in the rat gastrointestinal tract

Summary Gastrin and cholecystokinin (CCK) cells of the rat gastrointestinal tract have been studied by immunocytochemistry and radioimmunoanalysis. With antisera directed against the COOH-terminal tetrapeptide sequence, which is common to gastrin and CCK, three distinct endocrine cell types are detected. One of the cell types predominates in the antrum, is scarce in the rest of the gut and corresponds to the gastrin cell. The second cell type is virtually confined to the duodenum and jejunum and corresponds to the CCK cell. The third cell type occurs disseminated in the small intestines, predominates in the ileum, and reacts with COOH-terminus-specific antisera only following diethylpyrocarbonate and not following formaldehyde fixation. It is possible that the third cell type stores a third member of the gastrin-CCK family of gut hormones.     

The reactivity of mononucleotides with cholecystokinin/gastrin antisera

Dibutyryl cyclic GMP has been reported to interact with antisera specific for C-terminal tetrapeptide amide common for cholecystokinin (CCK) and gastrin. Moreover, cyclic nucleotides elute by gel chromatography in the same position as the free CCK/gastrin tetrapeptide. Therefore, we have examined the reactivity of 25 mononucleotides with eight CCK and gastrin antisera. The results show that the nucleotides all bind poorly to the antisera (nucleotide concentration required 1 mM). Hence, endogenous cyclic nucleotides, which are present in biological extracts in pM to nM concentrations, do not interfere with immunochemical CCK or gastrin measurements. The antisera displayed highly individual patterns of reactivity without preferential binding of di- or monobutyryl cyclic nucleotides (AMP, GMP or IMP). Thus, the present results do not support the idea of structural resemblance between the C-terminus of CCK/gastrin peptides and butyryl derivatives of cyclic GMP. Enzymatic treatment of the antral tetrapeptide-like immunoreactivity showed that nucleotides do not contribute to this material, which appears exclusively peptidergic.     

Cellular origins of different forms of gastrin. The specific immunocytochemical localization of related peptides.

We have localized the antigenic determinants for the main forms of gastrin (big gastrin, G34, and little gastrin, G17) in hog antral mucosa using sequence specific antibodies and an indirect immunofluorescence technique. Populations of monospecific antibodies were obtained after affinity immunoadsorption to remove populations of unwanted specificity. The specificity of the purified antisera was established by direct binding of 125I labeled peptides to antisera at the same dilutions as those used in immunocytochemistry. The results indicate that in hog antral mucosa there is a single population of cells with the antigenic determinants of the C-terminal region of G17 and G34, the N-terminal region of G17, the N-terminal region of G34, and the intact G17 molecule. In duodenum there are cells with only C-terminal reactivity; since gastrin and CCK share a common C-terminal sequence it is concluded that this cell type contains CCK-like peptides rather than gastrin.     

Isolation from chicken antrum, and primary amino acid sequence of a novel 36-residue peptide of the gastrin/CCK family

A peptide that cross-reacted with C-terminal gastrin/CCK antisera was isolated from chicken antral extracts by a combination of gel filtration and reversed-phase HPLC. The sequence was: Phe-Leu-Pro-His- Val-Phe-Ala-Glu-Leu-Ser-Asp-Arg-Lys-Gly-Phe-Val-Gln-Gly-Asn-Gly-Ala- Val-Glu-Ala-Leu-His-Asp-His-Phe-Tyr-Pro-Asp-Trp-Met-Asp-Phe(NH2). Aside from the C-terminal tetrapeptide and the Tyr residue, the molecule does not resemble other known forms of gastrin or CCK. The peptide was a potent stimulus of avian gastric acid but not pancreatic secretion. The results have important implications for the structure-activity and evolutionary relationships of the gastrin/CCK family.     

Identification of gastrin and multiple cholecystokinin genes in teleost

To identify the teleost gastrin, CCK/gastrin family genes were isolated from puffer and flounder. The cDNA of puffer gastrin, CCK1 and CCK2 were 678, 752 and 533bp, respectively. Puffer gastrin gene consists of three exons, in contrast, CCKs consist of four exons. Flounder gastrin mRNA (526bp) was expressed in the intestine but not in the brain. It was developed synchronously with the stomach differentiation in the larval stage. The phylogenetic analysis shows that puffer and flounder gastrin classified into the vertebrate gastrin cluster and two types of CCK were probably produced by the genome duplication occurred in teleost phylogeny.     

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.     

Abnormal gastric morphology and function in CCK-B/gastrin receptor-deficient mice.

Mice lacking the cholecystokinin (CCK)-B/gastrin receptor have been generated by targeted gene disruption. The roles of this receptor in controlling gastric acid secretion and gastric mucosal growth have been assessed. The analysis of homozygous mutant mice vs. wild type included measurement of basal gastric pH, plasma gastrin concentrations as well as quantification of gastric mucosal cell types by immunohistochemistry. Mutant mice exhibited a marked increase in basal gastric pH (from 3.2 to 5.2) and about a 10-fold elevation in circulating carboxyamidated gastrin compared with wild-type controls. Histologic analysis revealed a decrease in both parietal and enterochromaffin-like (ECL) cells, thus explaining the reduction in acid output. Consistent with the elevation in circulating gastrin, antral gastrin cells were increased in number while somatostatin cells were decreased. These data support the importance of the CCK-B/gastrin receptor in maintaining the normal cellular composition and function of the gastric mucosa.     

Development of region-specific antisera for the C-terminal tetrapeptide of gastrin/cholecystokinin and their use in studies of immunoreactive forms of cholecystokinin in rat brain

Molecular forms of cholecystokinin in rat brain were studied by radioimmunoassay using two new antisera raised against the C-terminal tetrapeptide common to cholecystokinin and gastrin. Evidence is presented to show that one antiserum (L112) reacts at the C-terminus of the tetrapeptide, while the other antiserum (L131) reacts at its N-terminus. With antiserum L112 the predominant immunoreactive form of CCK found in extracts of rat brain corresponded to the C-terminal octapeptide; a minor immunoreactive form eluted from Sephadex G25 between the C-terminal octapeptide and the tetrapeptide. A similar pattern of molecular forms was found using a third antiserum (L48) previously shown to react well with the C-terminal octapeptide and poorly with the C-terminal tetrapeptide. Antisera L112 and L48 also revealed a quantitatively similar distribution of immunoreactive material in different regions of rat and cow brain. In contrast, antiserum L131 failed to demonstrate significant amounts of immunoreactive material in rat brain. It is concluded that the C-terminal octapeptide of cholecystokinin predominates in rat brain and that contrary to findings of previous workers there is little or no free C-terminal tetrapeptide present.     

Implication of gastrin in cyclooxygenase-2 expression in Helicobacter pylori infected gastric ulceration

Gastroduodenal ulcerations have worldwide distribution and the infection with Helicobacter pylori (HP) has been implicated in pathogenesis of this disease. The HP infection is usually accompanied by hypergastrinemia and enhanced generation of prostaglandins (PG), both implicated in the pathogenesis of peptic ulcerations but no study has been undertaken to assess the relationship between the HP infection and coexpression of gastrin and cyclooxygenases (COX), the rate limiting enzymes in the PG production. Since HP infection, usually accompanying peptic ulcerations, results in increased release of gastrin, a potent gastric mitogen that might be capable to induce COX-2 and to generate PG, we decided 1) to compare the seroprevalence of HP and its cytotoxic protein, CagA, in gastric ulcer patients with those in age- and gender-matched controls; 2) to determine the gene expression of gastrin and its receptors (CCK(B)-R) at the margin of gastric ulcer and in the mucosa of antrum and corpus before and after successful eradication of HP, 3) to assess the plasma levels and gastric luminal contents of gastrin before and after HP eradication and 4) to examine the mRNA and enzyme protein expression of COX-1 and COX-2 as well as the PGE2 generation in ulcer margin tissue and gastric antral and fundic mucosa before and after the HP eradication. The trial material included 20 patients with gastric ulcer and 40 age- and gender-matched controls. Anti-HP and anti-CagA IgG seroprevalence was estimated by specific antisera using ELISA tests. Gene expressions of gastrin, CCK(B)-R, COX-1 and COX-2 were examined using RT-PCR with beta-actin as a reference and employing Western blotting for COX-2 expression, while gastrin and PGE2 were measured by RIA. All gastric ulcers were located at smaller curvature within the antral mucosal area. The seroprevalence of HP, especially that expressing CagA, was significantly higher in gastric ulcers (85%) than in controls (62.5%). Both gastrin and CCK(B)-R mRNA were detected by RT-PCR in ulcer margin and gastrin mRNA was overexpressed in remaining antral mucosa, while CCK(B)-R mRNA was overexpressed in fundic mucosa of HP infected patients. Similarly, COX-2 mRNA and protein were found in margin of gastric ulcer and in the HP infected antral and fundic mucosa but not in the mucosa of HP eradicated patients in whom ulcers completely healed and gastrin was expressed only in antrum, CCK(B)-R only in corpus, while COX-1 was detected both in antrum and corpus. HP positive gastric ulcer patients showed about three times higher levels of plasma immunoreactive gastrin and about 50% higher luminal gastrin contents than the HP negative controls and this increased plasma and luminal gastrin was normalized following the HP eradication. A significant fall in gastrin and CCK(B)-R mRNA expression was noticed six weeks after HP eradication in gastric antral and fundic mucosa, while COX-2 mRNA completely disappeared after this treatment. We conclude that 1) HP infected gastric ulcer margin coexpresses gastrin, its receptors (CCK(B)-R), and COX-2; 2) HP infection may be implicated in gastric ulceration via increased release of gastrin that could be responsible for the overexpression of COX-2 that in turn could help ulcer healing through the stimulation of mucosal cell growth, restoration of the glandular structure and angiogenesis in the ulcer area and 3) gastrin produced in HP infected antral mucosa seems to be involved in the induction of COX-2 and PG production by this enzyme and this may contribute to the ulcer healing.     

Localization of a peptide identified by antibodies to gastrin/CCK in the gut of Cancer magister

Summary A gastric peptide from the Dungeness crab (Cancer magister), extracted and characterized previously (Larson and Vigna 1983b), was localized in the foregut (stomach) of this species by immunocytochemistry using antisera specific for the bioactive carboxy-terminal amino acid sequence common to gastrins and cholecystokinins (CCKs). Immunoreactivity was found in all gastric epithelial cells and in the procuticle. Electron microscopy revealed an absence of peptidergic secretory granules in the gastric epithelial cells. The pattern of immunostaining suggests that the gastric epithelial cells secrete this peptide apically where it is incorporated into the cuticle lining the lumen. Specific immunostaining could not be demonstrated in various neural ganglia or in the hypodermis. The distribution of this peptide is different from that of gastrin/CCK in vertebrates and other invertebrates. This suggests that the crab gastric peptide is sufficiently similar to gastrin/CCK to react with C-terminal specific antisera, but may be anatomically, functionally, and possibly phylogenetically otherwise unrelated.     

Importance of sulfation of gastrin or cholecystokinin (CCK) on affinity for gastrin and CCK receptors

We investigated the importance of sulfation of gastrin or cholecystokinin (CCK) on influencing their affinity for gastrin or CCK receptors by comparing the abilities of sulfated gastrin-17 (gastrin-17-II), desulfated gastrin-17 (gastrin-17-I), CCK-8 and desulfated CCK-8 [des(SO3)CCK-8] to interact with CCK or gastrin receptors on guinea pig pancreatic acini. For inhibiting binding of 125I-gastrin to gastrin receptors, gastrin-17-II (Kd 0.08 nM) greater than CCK-8 (Kd 0.4 nM) greater than gastrin-17-I (Kd 1.5 nM) greater than des(SO3)CCK-8 (Kd 28 nM). For inhibiting binding of 125I-Bolton Hunter-labeled CCK-8 to CCK receptors the relative potencies were: CCK-8 much greater than des(SO3)CCK-8 = gastrin-17-II greater than gastrin-17-I. Each peptide interacted with both high and low affinity CCK binding sites. The relative abilities of each peptide to interact with high affinity CCK receptors showed a close correlation with their abilities to cause half-maximal stimulation of enzyme secretion. These results demonstrate that, in contrast to older studies, sulfation of both CCK and gastrin increase their affinities for both gastrin and CCK receptors. Moreover, the gastrin receptor is relatively insensitive to the position of the sulfate moiety, whereas the CCK receptor is extremely sensitive to both the presence and exact position of the sulfate moiety.     

Immunohistochemical evidence of gastro-entero-pancreatic neurohormonal peptides of vertebrate type in the nervous system of the larva of a dipteran insect, the hoverfly, Eristalis aeneus

Using rabbit and guinea-pig antisera, raised against GEP neurohormonal peptides of mammalian origin, cells were observed in the brain and/or in the fused ventral ganglia of the last (fifth) larval instar of the hoverfly, Eristalis aeneus, being immunoreactive with antisera against insulin, somatostatin, glucagon, PP, secretin, gastrin/CCK/caerulein; substance P, enkephalin and endorphin. Most of these GEP neurohormonal peptides also occurred in nerve fibers. No immunoreactive cells or nerve fibers could be detected with antisera against GIP, VIP, (the central fragments of) CCK, bombesin or neurotensin. The antisera tested failed to reveal any immunoreactive cells or nerves in Weismann's ring (fused corpus allatum/corpus cardiacum and thoracic gland) or in different parts of the alimentary tract. The observations support the hypothesis that neuronal GEP hormonal peptide production in the brain is a genuinely original mechanism and the appearance of endocrine cells in the gut a later feature in evolution.     

Interrelationships between circulating gastrin and iron status in mice and humans

The observations that the peptide hormone gastrin interacts with transferrin in vitro and that circulating gastrin concentrations are increased in the iron-loading disorder hemochromatosis suggest a possible link between gastrin and iron homeostasis. This study tested the hypothesis that gastrin and iron status are interrelated by measurement of iron homeostasis in mice and humans with abnormal circulating gastrin concentrations. Intestinal iron absorption was determined by (59)Fe uptake following oral gavage, and concentrations of duodenal divalent metal transporter-1 (DMT-1) and hepatic hepcidin mRNAs were determined by quantitative real-time PCR in agastrinemic (GasKO), hypergastrinemic cholecystokinin 2 receptor-deficient (CCK2RKO), or wild-type mice. Iron status was measured by standard methods in the same mice and in hypergastrinemic humans with multiple endocrine neoplasia type 1 (MEN-1). Iron absorption was increased sixfold and DMT-1 mRNA concentration fourfold, and transferrin saturation was reduced 0.8-fold and hepcidin mRNA expression 0.5-fold in juvenile GasKO mice compared with age-matched wild-type mice. In mature mice, few differences were observed between the strains. Juvenile CCK2RKO mice were hypergastrinemic and had a 5.4-fold higher DMT-1 mRNA concentration than wild-type mice without any increase in iron absorption. In contrast to juvenile GasKO mice, juvenile CCK2RKO mice had a 1.5-fold greater transferrin saturation, which was reflected in a twofold increase in liver iron deposition at maturity compared with wild-type mice. The correlation between transferrin saturation and circulating gastrin concentration observed in mutant mice was also observed in human patients with MEN, in whom hypergastrinemia correlated positively (P = 0.004) with an increased transferrin saturation. Our data indicate that, in juvenile animals when iron demand is high, circulating gastrin concentrations may alter iron status by a CCK2R-independent mechanism.     

Gut bitter taste receptor signalling induces ABCB1 through a mechanism involving CCK

T2Rs (bitter taste-sensing type 2 receptors) are expressed in the oral cavity to prevent ingestion of dietary toxins through taste avoidance. They are also expressed in other cell types, including gut enteroendocrine cells, where their physiological role is enigmatic. Previously, we proposed that T2R-dependent CCK (cholecystokinin) secretion from enteroendocrine cells limits absorption of dietary toxins, but an active mechanism was lacking. In the present study we show that T2R signalling activates ABCB1 (ATP-binding cassette B1) in intestinal cells through a CCK signalling mechanism. PTC (phenylthiocarbamide), an agonist for the T2R38 bitter receptor, increased ABCB1 expression in both intestinal cells and mouse intestine. PTC induction of ABCB1 was decreased by either T2R38 siRNA (small interfering RNA) or treatment with YM022, a gastrin receptor antagonist. Thus gut ABCB1 is regulated through signalling by CCK/gastrin released in response to PTC stimulation of T2R38 on enteroendocrine cells. We also show that PTC increases the efflux activity of ABCB1, suggesting that T2R signalling limits the absorption of bitter tasting/toxic substances through modulation of gut efflux membrane transporters.     

Postembryonic development of Arg-Phe-amide-like and cholecystokinin-like immunoreactive neurons in the blowfly optic lobe

Summary The adult optic lobes of the blowfly Calliphora erythrocephala were found to be innervated by more than 2000 neurons immunoreactive to antisera raised against the neuropeptides FMRFamide, its fragment RFamide, and gastrin/cholecystokinin (CCK). All of the CCK-like immunoreactive (CCK-IR) neurons also reacted with antisera to RFamide, FMRFamide and pancreatic polypeptide. A few RFamide/FMRFamide-like immunoreactive (RF-IR) neurons did not react with CCK antisera; they reacted instead with antisera to Leu-enkephalin and Met-enkephalin-Arg⁶-Phe⁷. The RF-IR neurons are, thus, heterogeneous with respect to their contents of immunoreactive peptides. Two of the RF-IR neuron types innervating the adult optic lobes could be traced in their entirety only after following their postembryonic development, because of the complexity of the trajectories of the immunoreactive neuronal process in the adult insect. The majority of the cell bodies of the RF-IR and CCK-IR neurons lie within the optic lobes and are derived from imaginal neuroblasts of the inner and outer optic anlagen. Six of the peptidergic neurons are, however, metamorphosing larval neurons with their cell bodies in the central part of the protocerebrum. The full extent of immunoreactivitiy is not attained in some of the neurons until the late pupal or early adult stage. The larval optic center was also found to be innervated by neurons immuno-reactive with both RFamide and CCK antisera. The cell bodies of these RF-IR/CCK-IR neurons are located near the developing lamina (one on each side). In the 24 h pupa, the cell bodies of these neurons are still immunoreactive, but thereafter they cannot be immunolabeled apparently due to cell death or a change in transmitter phenotype.