Identification and localization of material with gastrin-like immunoreactivity in the neural ganglion of a protochordate, Ciona intestinalis

Little is known of the identity of gastrin/cholecystokinin (CCK)-like peptides in protochordates. These animals are at a level of organization corresponding to that from which the vertebrate line arose; in order to shed light on the origins of gastrin/CCK-like peptides, we have studied by immunochemical methods these peptides in a protochordate, Ciona intestinalis. In radioimmunoassay, boiling water extracts of the neural ganglion reacted with C-terminal specific gastrin/CCK antibodies, but not N-terminal or intact G17 specific antibodies. Of particular importance was the fact that a gastrin antibody which reacts weakly with CCK8 showed full activity with the Ciona material, suggesting that it resembles the C-terminus of gastrin. A single major peak was found by gel filtration and HPLC. In immunohistochemistry, nerve cell bodies were found in the cortical regions of the ganglion, and abundant fibres ramified in the central neuropile. We conclude that peptides of the gastrin/CCK series occur in nervous tissue in protochordates, and that while they are distinguishable from known forms of both gastrin and CCK, they resemble C-terminal fragments of the mammalian gastrins.     

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.     

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.     

Receptors for cholecystokinin and gastrin peptides display specific binding properties and are structurally different in guinea-pig and dog pancreas

In the light of the strong potency of gastrin-related peptides on pancreatic exocrine secretion in dog, we analyzed the binding properties of peptides related to cholecystokinin (CCK) and gastrin on dog pancreatic acini compared to guinea-pig acini. Moreover, we determined apparent molecular masses of photoaffinity labelled CCK/gastrin receptors in the two models. Using the CCK radioligand, receptor selectivity towards CCK/gastrin agonists and antagonists was found to be lower in dog acini than in guinea-pig acini. Performing the binding with CCK and gastrin radioligands in combination with N2,O2'-dibutyryl-guanosine 3',5'-monophosphate, revealed that in dog acini there exist two different sub-classes of CCK/gastrin receptors having high and low selectivity, the latter ones being able to bind gastrin with high affinity (Kd = 2.1 nM). SDS-PAGE analysis of covalently cross-linked receptors using several photosensitive CCK and gastrin probes of different peptide chain lengths demonstrated that in guinea-pig, CCK peptides bound to a 84-kDa component whereas in dog pancreas, CCK and gastrin peptides bound to three distinct molecular species (Mr approximately equal to 78,000, 45,000, 28,000). Performing cross-linking in the presence of 1 microM CCK indicated that a 45-kDa protein is the putative CCK/gastrin receptor in dog pancreas. Our results support the concept of heterogeneity of CCK/gastrin receptors.     

Gastrin/cholecystokinin-like post-prandial variations: quantitative and qualitative changes in the haemolymph of penaeids (Crustacea Decapoda)

Summary Gastrin/cholecystokinin-like (G/CCK) peptides from the haemolymph of two penaeidsPenaeus japonicus andP. stylirostris (Crustacea, Decapoda) were characterized by gel filtration. In starved shrimps, four classes of peptides were found. One fraction elutes in the void volume, a second has a molecular weight around 5 kDa, a third between 1 and 2.5 kDa, and the fourth fraction is composed of very small peptides. Differences exist between the two species concerning the relative proportion of each molecule.After a meal, there is a two-fold increase of total G/CCK-like peptides and marked qualitative changes are described. Briefly, high molecular weight fractions decrease while low molecular ones increase. After 5 h a return to the initial concentrations is observed. These changes are more pronounced inP. stylirostris. Such variations suggests a role for these peptides in regulation of crustacean digestive processes.     

Identification of ostrich and chicken cholecystokinin cDNA and intestinal peptides

The cDNAs encoding the preprohormones of the regulatory peptides cholecystokinin (CCK) and the related gastrin have been identified in a number of vertebrate species. However, from birds only chicken preprogastrin is known. In the present study preproCCK cDNA was identified in two species of birds, ostrich and chicken. In addition, the molecular forms of the bioactive peptides expressed in the small intestine were characterized. Both preproCCKs contain mono basic processing sites for the production of CCK-70 and -8 as seen in turtle and bullfrog. However, compared to these species an unusually large proportion was processed to the small forms CCK-7 and -8 and only minute amounts to larger forms. The encoded preprohormones are very similar to each other and to turtle CCK. Furthermore, they also show a high degree of similarity to the CCKs identified in more distant vertebrates. This confirms that CCK is highly conserved among vertebrates while the structure of gastrin, the other member of the CCK/gastrin family, is considerably more variable.     

Gastrin/cholecystokinin-like immunoreactive peptides in the Dungeness crab, Cancer magister (Dana): immunochemical and biological characterization

The purpose of this investigation was to characterize a gastrin/cholecystokinin-like immunoreactant (G/CCK-LI) extractable from the crab, Cancer magister. G/CCK-LI was extracted best in boiling water and was found mainly in the stomach, hemolymph and carapace. A relatively large immunoreactive peptide in the stomach and apparently smaller forms in the hemolymph and carapace were separated by Sephadex G-50 fractionation. Anion-exchange chromatography further fractionated the stomach form into three major peaks. The crab material cross-reacted with three antisera specific for the common C-terminus of gastrin/CCK, but cross-reacted much less with three antisera directed against other portions of the gastrin molecule. Partially purified crab stomach G/CCK-LI inhibited the binding of labeled CCK to mouse brain G/CCK receptors but not to rat pancreatic CCK receptors. The crab peptide did not stimulate rat gastric acid or rat pancreatic amylase secretion. These results indicate that the crab peptides are structurally similar to, but distinguishable from, the bioactive C-terminal amino acid sequence common to gastrins and CCKs.     

Chromatographic characterization of gastrin/cholecystokinin peptides in bovine and porcine pituitary

Gastrin/CCK peptides in extracts from bovine and porcine pituitary have been characterized by Sephadex gel filtration, reverse phase liquid chromatography and a CCK radioimmunoassay (RIA) which detects both CCK and gastrin [4]. Porcine pituitary extracts contain a small amount of two peptides with chromatographic behavior similar to porcine antral gastrins. However, bovine pituitaries lack gastrin and contain instead substantial quantities of a peptide which co-elutes with CCK8 sulfate. We have previously shown that rat pituitary also contains CCK8 sulfate-like peptides but lacks gastrin [3]. It is clear from this work that species differences exist in the gastrin/CCK pituitary peptides. This points out the necessity of a careful chemical characterization of pituitary gastrin/CCK peptides in any species prior to physiological or pharmacological experimentation.     

Long-term comparative effect of cholecystokinin and gastrin on mouse stomach, antrum, intestine, and exocrine pancreas

Mice were injected three times a day for 12 days with 300 micrograms/kg body weight of gastrin G17 or 37.5 Ivy dog U/kg body weight of CCK or saline. Other mice were also injected four times an hr for 1 hr with 7.5 micrograms/kg of gastrin, nine Ivy dog U/kg of CCK or saline; 1 hr before killing, they were injected with tritiated thymidine to evaluate the labelling indices in peptic, antral, duodenal, jejunal, and ileal mucosae. Four hours after the first injection of the two peptides, the peptic labelling indices increased while those of intestinal mucosa increased 8 hr after these injections. Long-term injections of CCK had a trophic effect on secretory cells of the digestive tract: the number of gastric zymogenic cells, Paneth cells, and the mucous cells of Brünner glands were hypertrophied. The pepsin, amylase, chymotrypsin, and lysozyme activities increased in stomach, exocrine pancreas, and intestine, respectively. Neither parietal cells nor intestinal enterocytes and hydrolase activities were affected. The trophic effect of long-term injections of gastrin is confirmed on parietal cells and exocrine pancreatic parenchyma and is demonstrated in Paneth cells. Confirming cytological results, pancreatic lipase and amylase activities and intestinal lysozyme activity were increased after gastrin. Although CCK and gastrin have a structural analogy, these two peptides did not affect the same cellular types. A specific action of CCK on the main secretory cells of the digestive mucosa is demonstrated.     

Sulfakinins reduce food intake in the desert locust, Schistocerca gregaria

In vertebrates, the peptides cholecystokinin (CCK), neuropeptide Y, galanin, and bombesin are known to be involved in the control of food intake. We report here that insect sulfakinins, peptides which display substantial sequence similarities with the vertebrate gastrin/CCK peptide family, significantly inhibit food uptake in fifth instar nymphs of the locust, Schistocerca gregaria. Upon injection of Lom-sulfakinin, a neuropeptide present in the corpus cardiacum of locusts, food intake was significantly reduced in a dose-dependent manner within a fixed 20 min time period. The induced effect ranged from 13% inhibition (10 pmol of injected peptide) to over 50% inhibition at 1 nmol. Other naturally occurring sulfakinins from different insect species also elicited this satiety effect. Analogous to the satiety effect of CCK in vertebrates, the sulfate group is required for activity. No effect on the palptip resistance was found after injection with sulfakinin. Therefore it seems unlikly that sulfakinins reduce food intake by decreasing the sensitivity of the taste receptors.     

Effects of exogenous cholecystokinin and gastrin on the secretion of trypsin and chymotrypsin from yellowtail (Seriola quinqueradiata) isolated pyloric caeca

The humoral control of secretion of the proteolytic enzymes trypsin and chymotrypsin was studied in yellowtail (Seriola quinqueradiata). In vitro trials were performed to investigate the effects of cholecystokinin (CCK) and two commercially available gastrin peptides. Isolated preparations of pyloric caeca/pancreas release trypsin and chymotrypsin when incubated with cholecystokinin (CCK) at 10 microM and gastrin I (G1) at 50 microM after 15 min of incubation. On the other hand, G1 at 10 microM and gastrin-related peptide (G2) did not enhance trypsin and chymotrypsin secretion. The studies concerning the CCK effects at different incubation temperatures have shown that trypsin and chymotrypsin secretion at 25 degrees C was stimulated by CCK after 15 min, while at 10, 15 and 20 degrees C the stimulatory effects of CCK were observed only after 30 min of incubation. The CCK effects were increased at higher incubation temperatures and longer incubation periods.     

Relationship of CCK/gastrin receptor binding to amylase release in dog pancreatic acini

Effects of synthetic peptides belonging to the CCK/gastrin family (CCK-39, CCK-8, G/CCK-4, G-17ns) on amylase release in dog pancreatic acini have been measured and correlated with binding of three radio-labelled CCK/gastrin peptides: ¹²⁵I-BH-(Thr,Nle)-CCK-9, ¹²⁵I-BH-(2–17)G-17ns and ¹²⁵I-BH-G/CCK-4 prepared by conjugation of the peptides to iodinated Bolton-Hunter reagent and purified by reverse-phase-HPLC. All the CCK/gastrin peptides produced the same maximal amylase release response. Half-maximal responses (D₅₀) were obtained with 2 · 10^?10 M CCK-8; 6 · 10^?10 M CCK-39; 10^?7 M G.17 ns and 2 · 10^?6 M G/CCK-4. Dose-response curves for G-17 ns and G/CCK-4 were similar in configuration but not parallel with those for CCK-8 and CCK-39.Binding studies with ¹²⁵I-BH(Thr,Nle)-CCK-9 demonstrated the presence of specific CCK receptors on dog pancreatic acini. There was a good correlation between receptor occupancy by CCK-8 and CCK-39 and amylase stimulation since maximal amylase stimulation was achieved when 40–50% of high affinity receptors were occupied. In contrast, a saturation of these receptors was required for maximal stimulation by G-17 ns and G/CCK-4 suggesting the existence of a fraction of receptors that can be occupied by G-17 ns and G/CCK-4 without stimulation of amylase release. Binding studies with labelled (2–17)-G-17 ns and G/CCK-4 confirmed the presence of high affinity sites for G-17 ns and G/CCK-4. These sites were not related to amylase release.This study points out a possible species specificity of biological action of gastrin/CCK peptides on pancreatic exocrine secretion in higher mammals.     

Is caerulein amphibian CCK?

The amphibian skin decapeptide caerulein is structurally related to the mammalian peptides gastrin and CCK, suggesting that the peptides might share a common evolutionary history. It has been suggested that caerulein is the amphibian counterpart of gastrin and CCK, and that the Amphibia do not possess authentic gastric and CCK. High Performance Liquid Chromatography (HPLC) in conjunction with radioimmunoassay using a caerulein-specific antiserum and C-terminal CCK antisera, was used to characterize CCK-and caerulein-like peptides in amphibian brain and gut. In the brain of Xenopus laevis, two CCK-like peptides were present, one of which was indistinguishable by HPLC from mammalian CCK8. No decapeptide caerulein was detected in the brain of Xenopus laevis or Rana temporaria. In the stomach of Xenopus and in the intestine of both species studied, CCK-like and caerulein-like peptides were present. The results indicate therefore that the Amphibia possess CCK8-like rather than caerulein-like peptides in brain. In contrast, stomach and intestine contain both CCK-like and caerulein-like peptides, but the latter are however distinguishable from the decapeptide found in skin.     

Isolation,identification, and synthesis of a disulfated sulfakinin from the central nervous system of an arthropods the white shrimp Litopenaeus vannamei

Two myotropic peptides displaying tyrosyl sulfation have been isolated from an extract of central nervous systems (brain, suboesophageal ganglion, thoracic ganglia, and ventral nerve cord) of the white shrimp Litopenaeus vannamei. Both peptides were identified by mass spectrometry and belong to the sulfakinin family of neuropeptides, which are characterized by the C-terminal hexapeptide Y(SO(3)H)GHMRF-NH(2) preceded by two acidic amino acid residues. Pev-SK 1 (AGGSGGVGGEY(SO(3)H)DDY(SO(3)H)GH(L/I) RF-NH(2)) has two sulfated tyrosyl residues and a unique (L/I) for M substitution in the C-terminal sequence. Pev-SK 2 (pQFDEY(SO(3)H)GHMRF-NH(2)) fully complies with the typical sulfakinin core sequence and is blocked by a pyroglutamyl residue. Synthetic analogs (sulfated and unsulfated) were synthesized and the tyrosyl sulfations were confirmed by myotropic activity studies and co-elution with the native fractions. Pev-SK 1 is the first disulfated neuropeptide elucidated in the phylum of the arthropoda, with the only other reported disulfated neuropeptide, called cionin, found in a protochordate. The similarities in amino acid sequence and posttranslational modifications of the crustacean sulfakinins and protochordate cionin provide further evidence for the hypothesis stating that gastrin/CCK, cionin, and sulfakinins originate from a common ancestral gastrin/CCK-like peptide.     

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.     

Molecular heterogeneity of canine cholecystokinin in portal and peripheral plasma

The release of molecular forms of cholecystokinin (CCK) into the portal and peripheral blood in response to an intraduodenal perfusion of sodium oleate (9 mmol X h-1) was studied in six conscious dogs with chronic portal vein catheters. Immunoreactive CCK as concentrated from 20 ml plasma by C18 SEP PAK cartridges and the pattern of molecular forms of CCK were studied by G50 gel filtration. CCK-like immunoreactivity (CCK-LI) was measured in the column eluates with antibody 5135, which measures gastrin and CCK equally and requires the intact carboxyl-terminus for full recognition. Gastrin was measured specifically with antibody 1611. Intraduodenal perfusion with oleate did not alter basal gastrin release. Release of CCK-LI by intraduodenal oleate was calculated by the increments of the integrated CCK-LI peaks over basal. Total CCK-like immunoreactivity (CCK-LI), calculated by integration of all CCK-LI peaks in gel filtration eluates, increased over basal by 12 fmol/ml in the portal and by 6 fmol/ml in the peripheral plasma after intraduodenal perfusion with sodium oleate. The main molecular forms eluted on gel filtration in positions of CCK33,39 and of CCK8. The pattern of CCK in the peripheral plasma was similar to that in the portal plasma except that in the peripheral plasma large molecular forms were more abundant than small forms. This finding was confirmed when CCK39 and CCK8 were infused either into the portal vein or into the peripheral vein and peripheral plasma CCK levels were measured. Elimination of CCK8 after portal vein infusion compared to peripheral vein infusion was about 3 times higher than that of CCK39. The abundance of large molecular forms of CCK in the circulating blood which are similar in potency to small forms, underlines their role in the physiology of CCK.     

Immunoreactive cholecystokinin in human and rat plasma: correlation of pancreatic secretion in response to CCK

Immunoreactive cholecystokinin (CCK) levels in human and rat plasma are described using a radioimmunoassay specific for the biologically active sulfated end of CCK. This assay detected significant changes in plasma cholecystokinin levels during intrajejunal administration of amino acids and intravenous infusions of CCK-8 which were followed by increased pancreatic secretion. In humans, the concentration (pg/ml) of plasma cholecystokinin increased from 10.8 to 18.9 following intrajejunal amino acid instillation and from 15.4 to 31.1 during CCK infusion, while pancreatic trypsin secretion increased more than 15 fold. Ingestion of a test meal also caused a rapid and significant elevation (P less than 0.05) in both plasma CCK (14.5-21.7 pg/ml) and gastrin (50-160 pg/ml) levels. In the rat, an injection of 46 ng of CCK-8 produced a 300% increase in immunoreactive plasma CCK levels (2 min) and caused peak pancreatic protein secretion within 5 min; 4 fold lower doses (11.5 ng) elevated plasma CCK by 38% and pancreatic protein secretion to a small but significant extent. The ability of this assay to detect various forms of sulfated CCK in human plasma was also determined. Following gel chromatography on Sephadex G-50, at least three different immunoreactive peaks were found in plasma from fasted subjects and after intrajejunal amino acid stimulation. While the lower molecular weight CCK peptides (CCK-8 and CCK-12) were detected in plasma from both fasted and stimulated subjects, the larger form (CCK-33) was only present in measurable concentrations after amino acid infusion. The simultaneous measurement of increased plasma CCK levels and pancreatic secretion and the changes in the distribution of CCK peptides following amino acid infusion provides strong support that this assay detects physiologically relevant changes in biologically active CCK peptides.     

Development of cholecystokinin radioimmunoassay using synthetic CCK-10 as immunogen

Using an antiserum generated against synthetic CCK-10, we have developed a radioimmunoassay specific for the carboxyl-terminus of cholecystokinin (CCK). Three rabbits were immunized with synthetic sulfated carboxy-terminal CCK decapeptide (CCK-10) conjugated to keyhole limpet hemocyanin. Using 125I-CCK-39 prepared by the Iodogen method as a tracer, we found that all immunized rabbits produced antibodies against the conjugate. Antiserum R016 had the highest titer (1:225,000 after four immunizations) and was studied most extensively. R016 recognizes all molecular forms of CCK, including unsulfated and oxidized forms, but has negligible cross-reactivity with gastrin and other peptides. Using CCK-8 as a standard, the assay has a minimum detection limit of 0.5 pM and an ED50 of 11.5 pM. Serial dilutions of water/acid extracts of canine intestine were parallel to serial dilutions of sulfated CCK-8, CCK-33 and CCK-39. The assay was used to measure CCK concentrations in canine plasma after C18 Sep-Pak extraction; the concentration of immunoreactive CCK increased from a basal value of 7.8 +/- 1.0 to 9.5 +/- 1.2 and 11.1 +/- 1.2 pM 30 and 60 min postprandially (P less than 0.05 by paired analysis). This sensitive and uniquely specific CCK radioimmunoassay should be useful in characterizing several aspects of CCK physiology and the method for generating CCK antisera should be of value to other investigators.     

Degradation of human gastrin and CCK by endopeptidase 24.11: differential behaviour of the sulphated and unsulphated peptides

The degradation of human sulphated heptadecapeptide gastrin (G17s) by human endopeptidase 24.11 was studied in vitro. The products of degradation were characterized by HPLC, region-specific gastrin radioimmunoassay and amino acid analysis. The enzyme cleaved G17s at four sites, Trp4-Leu5, Ala11-Tyr12, Gly13-Trp14 and Asp16-Phe17. The patterns of fragments produced when sulphated and unsulphated G17s are hydrolysed by endopeptidase 24.11 indicate that the enzyme cleaves both substrates at the same four bonds. However, the sulphated G17 was 3-times less rapidly degraded than the unsulphated G17 (G17ns). In contrast, the rate of cleavage of the octapeptide cholecystokinin (CCK8) was faster when the peptide was sulphated. The kinetic data of endopeptidase 24.11 indicated similar Km values for sulphated or unsulphated gastrin and CCK; sulphated CCK8 exhibited a 2-fold higher kcat/Km value compared to unsulphated CCK8, whereas G17s exhibited a 2-fold lower kcat/Km value compared to G17ns. The results indicate that the presence of a sulphate group causes a marked reduction in the rate of hydrolysis of gastrin by endopeptidase 24.11, whereas sulphation enhances cholecystokinin degradation by the same enzyme. They also suggest that endopeptidase 24.11 may be responsible for the difference in metabolism of sulphated and unsulphated G17, previously observed in human circulation.     

Gastrin and cholecystokinin in the Eastern Grey kangaroo, Macropus giganteus giganteus

Gastrin and CCK are believed to have a common ancestor. The gastrin structure has probably evolved from CCK-like peptides at a stage later than the amphibians. To trace the evolution of gastrin and CCK we have determined their structures in an Australian marsupial, the Eastern Grey kangaroo. The brain CCK was identical to CCK-8 of most mammals. The larger form of kangaroo gastrin was a 33mer with the sequence pELHPQDLPHLMTDLSKKKGPWQEEDAAY(SO₃)GWMDF-NH₂. The 11 italic residues indicate differences from human gastrin. Gastrin-15 and gastrin-16 comprised about 70% of the total immunoreactivity and resulted from cleavage after the second and third residue, respectively, of the unusual tribasic cleavage site. The smaller forms of kangaroo gastrin differ from most other mammalian gastrins in that the N-termini are not blocked with a pyroGlu moiety. Unlike other gastrins, kangaroo gastrin is more than 95% sulfated. The present study indicates that the gastrin structure, as defined by having a Tyr at position 6 from the C-terminus, evolved before the marsupials diverged from the other mammals 130 million years ago.