Metabolism and acid secretory effect of sulfated and nonsulfated gastrin-6 in humans

The antral hormone gastrin is synthesized by processing progastrin into different peptides that stimulate gastric secretion. The effect on acid secretion depends mainly on the metabolic clearance rate of the peptides, but some of them may differ in potency and maximum acid output at similar concentrations in plasma. Sulfated and nonsulfated gastrin-6 are the smallest circulating bioactive gastrins in humans. Their effect and metabolism have now been investigated in nine normal subjects and compared with nonsulfated gastrin-17, a main product of progastrin. Maximum acid output after stimulation with gastrin-17, sulfated gastrin-6, and nonsulfated gastrin-6 were 28.3 +/- 2.0, 24.5 +/- 2.0 (P < 0.02), and 19.3 +/- 2. 3 (P < 0.05) mmol H(+)/50 min, respectively, and the corresponding EC(50) values were 43 +/- 6, 24 +/- 2 (P < 0.01), and 25 +/- 2 (not significant) pmol/l. The half-life of gastrin-17 was 5.3 +/- 0.3 min, the metabolic clearance rate (MCR) was 16.5 +/- 1.3 ml. kg(-1). min(-1), and the apparent volume of distribution (V(d)) was 124.3 +/- 9.6 ml/kg. The half-lives of sulfated and nonsulfated gastrin-6 were 2.1 +/- 0.3 and 1.9 +/- 0.3 min, the MCRs were 42.8 +/- 3.7 and 139.4 +/- 9.6 ml kg(-1) min(-1) (P < 0.01), and the V(d) were 139.0 +/- 30.5 and 392.0 +/- 81.6 (P < 0.01) ml kg(-1). All pharmacokinetic parameters differed significantly from gastrin-17 (P < 0.01). We conclude that gastrin 6 has a higher potency but a lower efficacy than gastrin-17. The efficacy of gastrin-6 is increased by tyrosine O-sulfation, which also enhances the protection against elimination.     

Sulfation of gastrin: Effect on immunoreactivity

The effect of sulfuric acid esterification of Tyr-12 in gastrin-17 on immunoreactivity was evaluated by the ability of seventeen antisera raised against non-sulfated gastrin-17 to bind sulfated gastrins in extracts of gastrinoma and antral tissue. Using non-sulfated Tyr-12 iodinated gastrin as tracer, and non-sulfated gastrin-17 as standard the antisera showed three different patterns of reactivity: Three antisera (Nos. 2602, 2605 and 4562) bound sulfated gastrins with low (4–23%) potency; four antisera (Nos. 2604, 2720, 4710 and 4713) measured sulfated gastrins with a potency similar to that of non-sulfated gastrins (81–100% crossreactivity); whereas ten antisera (Nos. 2601, 2606, 2609, 2716, 2717, 2718, 4556, 4559, 4560 and 4563) displayed enhanced reactivity with sulfated gastrins (130–373% cross-reactivity). Using Gly-2 iodinated gastrin as tracer, the latter type of antisera reacted almost equally with sulfated and non-sulfated gastrins, suggesting that the apparent increase in binding of sulfated gastrins rather is due to increased displacement of Tyr-12 iodinated gastrin. The results show that derivatization of amino acid residues greatly influences antibody binding.     

Stability of tyrosine sulfate in acidic solutions

Tyrosine O-sulfation is a posttranslational modification of secretory and membrane proteins transported through the Golgi apparatus, which is widespread among higher eukaryotes. O-Sulfated tyrosines are not immediately identified during sequencing of peptides and proteins, because the sulfate ester is acid labile and rapidly hydrolyses to tyrosine in strong acidic solutions. Little is known about the hydrolysis at mildly acidic solutions, which are used during several protein purification and analysis procedures. We have examined the stability of tyrosine sulfate using sulfated gastrin-17, caerulein, and drosulfokinin as models for tyrosine O-sulfated peptides. The peptides were incubated in acidic solutions in a pH range of 1 to 3 at different temperatures and time spans. Only marginal hydrolysis of gastrin-17 was observed in triflouroacetic acid at room temperature or below. Comparison of the acid hydrolysis of the three peptides showed that hydrolysis rate depends mainly on the primary amino acid composition of the peptide. The activation energy (E(a)) for the hydrolysis of sulfated gastrin-17 was found to be E(a)=98.7+/-5 kJ mol(-1). This study serves as a general reference for handling tyrosine sulfated peptides in aqueous acidic solutions. We conclude that tyrosine sulfate is more stable under normal protein purification conditions than previously assumed.     

Pituitary gastrins. Different processing in corticotrophs and melanotrophs

The anterior, intermediate, and neural lobes of porcine pituitaries contain different molecular forms of gastrin. In the anterior lobe, unsulfated large gastrins, component I, and gastrin-34 constitute the main forms. In contrast in the intermediate and neural lobes, gastrin-17, sulfated as well as unsulfated, predominates. Since component I and gastrin-34 are biosynthetic precursors of gastrin-17, the findings indicate that proteolytic processing and amino acid derivatization (sulfation) differs between the lobes. This difference, together with the localization of anterior lobe gastrin in corticotrophs and intermediate lobe gastrin in melanotrophs, emphasizes the close relation and parallelism of the biosynthesis of gastrin and corticotropin peptides.     

Alpha-carboxyamidation of antral progastrin. Relation to other post-translational modifications

Using radioimmunoassays for amidated and glycine-extended gastrin before and after trypsin-carboxypeptidase B cleavage and chromatography, alpha-carboxyamidation of porcine antral progastrin has been related to tyrosine-O-sulfation and proteolytic cleavages. Corresponding to the sequence at the proteolysis and amidation site, -Gly-Arg-Arg-, antrum contained three COOH-terminally extended precursor types. The glycine-extended gastrins were present in the highest concentrations (241 +/- 58 pmol/g). The degree of tyrosine-O-sulfation was identical for amidated and precursor gastrins irrespective of component size, whereas the component size differed for glycine-extended and amidated forms. For instance, gastrin-34-Gly constituted 54% of the glycine-extended gastrins, while gastrin-34 comprised 8% of the amidated gastrins. The results indicate that tyrosine-O-sulfation occurs prior to NH2-terminal cleavages, which again precede carboxyamidation; but a significant correlation between tyrosine-O-sulfation and proteolytic cleavages or alpha-carboxy-amidation of antral gastrin could not be demonstrated. Furthermore, our results suggest that the immediate precursor of the principal hormonal form, gastrin-17, is gastrin-17-Gly rather than gastrin-34 as previously believed.     

Identification of gastrin molecular variants in gastrinoma syndrome

The molecular species of gastrin in the circulation and in tumor extracts were studied in two groups of patients: (1) with benign gastrinoma and (2) with gastrinoma with liver metastases. Radioimmunoassays (RIAs) and immunoaffinity chromatography for the amino (NH2)- and amidated COOH-terminus of gastrin-17 (antiserum G17) and the NH2-terminus of gastrin-34 (antiserum G34) were employed. In both benign and metastatic tumors the molecular forms of gastrin in boiling water extracts measured by the gastrin-17 NH2- and COOH-terminal assays were similar. In addition to a molecular component resembling the amidated gastrin-17, there were also significant amounts of larger molecular weight (mol. wt.) forms. The larger mol. wt. forms absorbed by the NH2-terminus of G17 antiserum corresponded to the COOH-terminus-extended forms of gastrin-17. Furthermore, larger mol. wt. gastrins immunopurified by antiserum to the NH2-terminus of gastrin-34 corresponded to gastrin-34 extended molecules. Sera of patients with liver metastases had higher concentrations of the NH2-terminal of gastrin-17 whereas sera of patients with benign gastrinoma contained predominantly gastrins detected by the COOH-terminal assay. These results suggest that: (a) there are differences in the molecular pattern of gastrin in the circulation of patients with benign and metastatic gastrinomas; (b) gastrins which are fully processed with carboxy-terminal amidation predominate in the circulation of patients with benign gastrinoma; and (c) gastrins containing the gastrin-17 and COOH-terminally extended gastrin-17 and gastrin-34 precursor molecules occur in high concentration in the circulation of gastrinoma patients with metastases to the liver.     

Complete sulfation of jejunal gastrin in the human fetus

The degree of tyrosine-O-sulfation and the ratio between large (gastrin-34 and component I) and small (gastrin-17 and -14) molecular forms of gastrin were studied in extracts of human fetal (n = 14) and adult (n = 9) antrum, duodenum, jejunum and pancreas. Boiled water extracts were applied to gel- and ion-exchange chromatography before and after treatment with trypsin and arylsulfatase. The fractions were monitored with sequence-specific radioimmunoassays that distinguish sulfated from non-sulfated gastrins. In antrum and duodenum about half the gastrins were sulfated at all stages of development. In the fetal jejunum gastrin occurred in sulfated form only while in the adult 72% (range, 64–88%) of the jejunal gastrins were sulfated. The larger molecular forms of gastrin predominated in the fetal compared with the adult antrum. In duodenum and jejunum, however, the ratio between small and large forms was the same in fetus and adult. Gastrin was undetectable in both fetal and adult pancreas. The results show that the degree of sulfation of gastrin varies substantially in the different parts of the gut at different stages of development. The differences may have functional significance, since sulfation increases the pancreozyminic and cholecystokinetic potency of gastrin.     

Sulfated gastrin stimulates ghrelin and growth hormone release but inhibits insulin secretion in cattle

This study was designed to determine the effects of gastrin on the circulating levels of ghrelin, growth hormone (GH), insulin, glucagon and glucose in ruminants. Two experiments were done in eight Holstein steers. Animals were randomly assigned to receive intravenous bolus injections: (1) 0.1% bovine serum albumin in saline as vehicle, 0.8, 4.0 and 20.0 μg/kg body weight (BW) of bovine sulfated gastrin-34; (2) vehicle, 0.53 μg/kg BW of bovine sulfated gastrin-17 alone or combined with 20.0 μg/kg BW of [d-Lys³]-GHRP-6, the selective antagonist of GHS-R1a. Blood samples were collected from −10 to 150 min relative to injection time. Concentrations of acyl and total ghrelin in response to gastrin-34 injection were significantly increased in a dose-dependent manner. Concentrations of GH were also markedly elevated by gastrin-34 injection; however, the effect of 20.0 μg/kg was weaker than that of 4.0 μg/kg. The three doses of gastrin-34 equally decreased insulin levels within 15 min and maintained the level until the time of last sampling. Gastrin-34 had no effect (P > 0.05) on the levels of glucagon and glucose. Levels of acyl ghrelin increased after administration of gastrin-17 alone or combined with [d-Lys³]-GHRP-6; however, [d-Lys³]-GHRP-6 did not block the elevation of GH by gastrin-17. The present results indicate that sulfated gastrin stimulates both ghrelin and GH release, but the GHS-R1a may not contribute to the release of GH by gastrin. Moreover, sulfated gastrin seems to indirectly maintain the homeostasis of blood glucose through the down-regulation of insulin in ruminants.     

Naming progastrin-derived peptides

The antral hormone gastrin continues to be in focus, because its hormonal and growth promoting effects are essential both for the function of the normal stomach and for the pathogenesis of major dyspeptic and neoplastic diseases. Deduction of the progastrin structure has improved the insight in the cellular synthesis of gastrin, but has also revealed that the biosynthetic machinery is complex, and, accordingly, that progastrin is processed to a multitude of more or less bioactive fragments. The naming of these fragments has, however, become inconsistent and confusing. Therefore, we propose a systematic nomenclature for progastrin-derived peptides of which there are three classes: (I) The gastrins with the evolutionary preserved tetrapeptide amide (Trp-Met-Asp-PheNH₂) at the C-terminus, which ensures high-affinity binding to the gastrin (CCK-B) receptor. Among the gastrins, gastrin-34 and gastrin-17 constitute the primary forms. (II) Processing intermediates, which are early products of progastrin that contain the structure of the primary gastrins within their sequence, but still cannot bind the gastrin receptor due to insufficient processing at their C-terminus. (III) Flanking fragments from the N- and C-termini of progastrin that do not contain any primary gastrin in their sequence, but nevertheless may undergo posttranslational processing. Each fragment can be specified with suffixes corresponding to the derived sequence in progastrin.     

Rat progastrin processing yields peptides with altered potency at the CCK-B receptor

Details of prohormone processing patterns are revealed by purification and characterization of molecular forms stored in the tissues where the hormones are expressed. Molecular forms of rat gastrin were purified from antral extracts by gel permeation, anion exchange, and reverse-phase HPLC. Amidated and glycine-extended gastrins were detected with specific antisera and their structures determined by mass spectrometry. In rats, the only form shorter than gastrin-17 observed contained 16 amino acids. These data suggest that two enzymes process the amino terminus of gastrin-17. Pyrrolidone carboxylic acid peptidase removes the amino terminal pyrrolidone carboxylic acid (pyroGlu), forming gastrin-16. In mammals other than rat, gastrin-16 is then cleaved by dipeptidyl peptidase IV to form gastrin-14. In rat, this reaction does not take place because of proline residues Pro(2)-Pro(3)- in gastrin-16. Gastrin-16 is found in sulfated and nonsulfated forms and comprises 28% of the total gastrin immunoreactivity. Glycine-extended forms of gastrin-16 and gastrin-17 comprises 45% of the total gastrin immunoreactivity. The sulfated forms of gastrin-16 and gastrin-17 bind to the CCK-B receptor transfected into CHO cells with 10-fold higher affinity than the nonsulfated forms of these peptides. Therefore, processing of rat progastrin may modulate the expression of gastrin biological activity.     

The N-terminal tridecapeptide fragment of gastrin-17 inhibits gastric acid secretion

After a meal the serum concentrations of the N-terminal tridecapeptide-like fragment of gastrin-17, (1-13)G-17, increased markedly in patients with active duodenal ulcer, but less so in healthy subjects. Consequently the synthetic (1-13)G-17 was infused intravenously in doses that resulted in concentrations similar to those measured in duodenal ulcer patients in order to examine whether the N-terminal fragment influences gastric acid secretion. Doses of 125 and 400 pmol (1-13)G-17/kg per h inhibited the meal-stimulated acid secretion by 36% (P less than 0.05) and 66% (P less than 0.05) respectively. The release of endogenous C-terminal gastrin immunoreactivity was not influenced. The infusion of (1-13)G-17 also inhibited the acid response to exogenous gastrin-34, gastrin-17 and Peptavlon, but not to gastrin-4. The results suggest that the N-terminal gastrin-17 fragment--although devoid of the hitherto considered only active site of gastrin--plays a significant role in the regulation of the gastric acid secretion in patients with active duodenal ulcer.     

Complete tyrosine O-sulfation of gastrin in adult and neonatal cat pancreas

We have found gastrin in both the adult and neonatal cat pancreas. In contrast with the main production sites, antrum and duodenum, gastrin in the pancreas occurs in a single molecular form, tyrosine O-sulfated gastrin-17. Since tyrosine sulfation increases the pancreozymic effect of gastrin, the complete sulfation seems functionally expedient.     

Sequences of gastrins purified from a single antrum of dog and of goat

Heptadecapeptide gastrins (G17) have been purified and sequenced from a variety of species. However, progastrin (G34) sequences have been determined only for pig and human from purified peptides and for rat from cDNA. Since G34 in most species accounts for only approximately 5% of total antral gastrin, micropurification techniques must be employed to avoid the need for large quantities of antral tissue. Efficient purification methodology yielded 1.5 and 1.3 nmol of G34 from the antrum of a single goat and of a single dog, respectively. The N-terminal pyroglutamyl residues were enzymatically removed and the peptides were sequenced through to the proximity of their COOH-termini. The COOH-terminal sequences of goat and dog G34 were confirmed by sequencing the corresponding deblocked G17 from each animal. The previously published dog G17 sequence was shown to be incorrect. The sequences for dog and goat G34 are: Dog less than ELGLQGPPQLVADLSKKQGPWMEEEEAAYGWMDF# Goat less than ELGLQDPPHMVADLSKKQGPWVEEEEAAYGWMDF# Dog and goat gastrins differ in 3 sites in the 17 amino acid NH2-terminus and only a single site in G17 (the sites of differences are underlined). The ratio for sulfated to non-sulfated antral G17 is 9:1 for the goat and 1:9 for the dog.     

Metabolism of recombinant progastrin in sheep

Precursor forms of peptide hormones may be biologically active with effects distinct from the mature end product. Nonamidated progastrin-derived peptides stimulate growth of colonic epithelium and are elevated in the circulation of patients with colorectal carcinomas, whereas the amidated end product is the major regulator of gastric acidity. Using region-specific radioimmunoassays, we here compared the in vitro and in vivo metabolism of recombinant human progastrin-(6-80) and two other nonamidated gastrins, gastrin-17-Gly and Tyr(70)-progastrin-(71-80). Although progastrin-(6-80) was very stable in vitro, both progastrin-(6-80) and gastrin-17-Gly were degraded in vivo. The in vivo data were best fitted by a double-exponential decay curve, and the half-lives for progastrin-(6-80) (t1/2alpha = 5.1 +/- 1.1, t(1/2)beta = 42 +/- 11 min) were significantly (P < 0.05) longer than for gastrin-17-Gly (t(1/2)alpha = 2.2 +/- 0.6, t(1/2)beta = 13 +/- 1 min). Tyr(70)-progastrin-(71-80) was degraded more rapidly. Comparison with amidated gastrins suggests that peptide length, rather than sequence, is the critical determinant of clearance. Progastrin has the clearance characteristics to be considered a circulating hormone.     

Measurement of precursors for alpha-amidated hormones by radioimmunoassay of glycine-extended peptides after trypsin-carboxypeptidase B cleavage

Using fragment 5-17 of human gastrin-17 extended with glycine at the C-terminus as hapten, three of six rabbits produced high-titer, high-avidity antisera specific for glycine-extended gastrins. In combination with trypsin and carboxypeptidase B cleavage, radioimmunoassays based on these antisera measured progastrins in some extra-antral tissues and certain malignant tumors. The results show that sequential cleavage with trypsin and carboxypeptidase B followed by radioimmunoassay of glycine-extended peptides is a rapid and accurate procedure for measurement of biosynthetic precursors of alpha-amidated peptide hormones. Moreover, the procedure seems promising in the search for tumor markers.     

The metabolism of gastrin-52 and gastrin-6 in pigs

The kinetics and metabolism in various organs of three bioactive products of progastrin, the small sulfated and nonsulfated gastrin-6 and the large nonsulfated gastrin-52, were examined during intravenous administration in anesthetized pigs. The kidney, hindlimb, liver, head, and gut eliminated the hexapeptides efficiently, with a fractional extraction ranging from 0.50 to 0.28 (P<0.001-0.05). No metabolism was recorded in the lungs, and sulfation was without influence on the extraction of gastrin-6. Gastrin-52 was eliminated only in the kidney and the head, with a fractional extraction between 0.23 and 0.11 (P<0.01-0.05). The half-life of sulfated and nonsulfated gastrin-6 was 1.5+/-0.4 and 1.4+/-0.3 min, the metabolic clearance rate (MCR) was 80.8+/-7.6 and 116.0+/-13.5 ml x kg(-1) x min(-1) (P<0.05), and the apparent volume of distribution (V(dss)) was 199.3+/-70.1 and 231.4+/-37.3 ml/kg, respectively. The decay of gastrin-52 in plasma was biexponential. The half-lives of this biexponential after a bolus injection were 3.9+/-0.5 (T(1/2alpha)) and 25.7+/-1.4 (T(1/2beta)) min, and the MCR and V(dss) were 4.2+/-0.4 ml. kg(-1) x min(-1) and 116.2+/-16.2 ml/kg(1). We conclude that there is a differential elimination of progastrin products in splanchnic and nonsplanchnic tissue, which depends on the chain length of the peptides. Sulfation of gastrin-6 had no influence on the organ-specific extraction but reduced the MCR. Our results are in keeping with previous studies of nonsulfated gastrin-17, which is extracted in the kidney, head, limb, and gut but not in the liver.     

Preparation of tyrosine-O-[35S]sulfated cholecystokinin octapeptide from a nonsulfated precursor peptide

A rapid and simple one-pot method for O-sulfation of nonsulfated cholecystokinin octapeptide (CCK-8) was developed using sulfuric acid and dicyclohexylcarbodiimide (DCC) without protection of the amino acid side chains. The extent of sulfation was increased with increasing the amount of reactants, sulfuric acid, and DCC, and reached maximum (40%) with fourfold molar excess of sulfuric acid and 40-fold molar excess of DCC. The excess of nonsulfated peptide inhibited the sulfation. The sulfation product was purified by HPLC or TLC to give a pure sulfated substance which showed exactly the same behavior as that of an authentic O-sulfated CCK-8 on HPLC or TLC. The purified sulfated peptide was active in stimulating amylase secretion from rat pancreatic fragments, and amino acid analysis showed that the tyrosine residue in the peptide existed in O-sulfated form. Sulfation with [35S]sulfuric acid-DCC produced a radioactive substance, from which O-[35S]sulfated CCK-8 could be easily purified by two-dimensional TLC.     

Increased synthesis but decreased processing of neuronal proCCK in prohormone convertase 2 and 7B2 knockout animals

In addition to its role as a gut hormone, cholecystokinin (CCK) is a widespread and potent neurotransmitter. Its biosynthesis requires endoproteolytic cleavage of proCCK at several mono- and dibasic sites by subtilisin-like prohormone convertases (PCs). Of these, PC1 and PC2 are specific for neuroendocrine cells. We have now examined the role of PC2 and its binding protein, 7B2, in the neuronal processing of proCCK by measurement of precursor, processing-intermediates and bioactive end-products in brain extracts from PC2- and 7B2-null mice and from corresponding controls. PC2-null mice displayed a nine-fold increase of cerebral proCCK concentrations, and a two-fold increase in the concentrations of the processing-intermediate, glycine-extended CCK, whereas the concentrations of transmitter-active (i.e. alpha-amidated and O-sulfated) CCK peptides were reduced (61%). Chromatography showed that O-sulfated CCK-8 still is the predominant transmitter-active CCK in PC2-null brains, but that the fraction of intermediate-sized CCK-peptides (CCK-58, -33 and -22) was eight-fold increased. 7B2-null brains displayed a similar pattern but with less pronounced precursor accumulation. In contrast with the cerebral changes, PC2 deficiency was without effect on proCCK synthesis and processing in intestinal endocrine cells, whereas 7B2 deficiency halved the concentration of bioactive CCK in the intestine. The results show that PC2 plays a major neuron-specific role in the processing of proCCK.     

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.     

Molecular forms of gastrin in antral mucosa and serum of dogs.

Instillation of liver extract into antral pouches produced an increase in the serum concentrations of both little (G-17) and big (G-34) gastrins. The molar fraction of G-17 plus G-34 represented by G-17 was about 0.9 in antral mucosa and about 0.3 in serum 3 hr after initiating release with liver extract. The predominance of G-34 in serum can be accounted for only in part by its slower rate of removal from the blood so other factors probably also contribute. Although G-17 contributed only about 30% of the total molar concentration of gastrins in serum, it accounted for about 70% of the acid stimulatory activity because on a molar basis it is about five times more bioactive than G-34.