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.     

Interleukin-1beta up-regulates RGS4 through the canonical IKK2/IkappaBalpha/NF-kappaB pathway in rabbit colonic smooth muscle

Cellular synthesis of peptide hormones requires PCs (prohormone convertases) for the endoproteolysis of prohormones. Antral G-cells synthesize the most gastrin and express PC1/3, 2 and 5/6 in the rat and human. But the cleavage sites in progastrin for each PC have not been determined. Therefore, in the present study, we measured the concentrations of progastrin, processing intermediates and alpha-amidated gastrins in antral extracts from PC1/3-null mice and compared the results with those in mice lacking PC2 and wild-type controls. The expression of PCs was examined by immunocytochemistry and in situ hybridization of mouse G-cells. Finally, the in vitro effect of recombinant PC5/6 on progastrin and progastrin fragments containing the relevant dibasic cleavage sites was also examined. The results showed that mouse G-cells express PC1/3, 2 and 5/6. The concentration of progastrin in PC1/3-null mice was elevated 3-fold. Chromatography showed that cleavage of the Arg(36)Arg(37) and Arg(73)Arg(74) sites were grossly decreased. Accordingly, the concentrations of progastrin products were markedly reduced, alpha-amidated gastrins (-34 and -17) being 25% of normal. Lack of PC1/3 was without effect on the third dibasic site (Lys(53)Lys(54)), which is the only processing site for PC2. Recombinant PC5/6 did not cleave any of the dibasic processing sites in progastrin and fragments containing the relevant dibasic processing sites. The complementary cleavages of PC1/3 and 2, however, suffice to explain most of the normal endoproteolysis of progastrin. Moreover, the results show that PCs react differently to the same dibasic sequences, suggesting that additional structural factors modulate the substrate specificity.     

Mice overexpressing progastrin are predisposed for developing aberrant colonic crypt foci in response to AOM

Recent studies show that nonamidated gastrins (Gly-gastrin and progastrin) stimulate colonic proliferation. However, the role of nonamidated vs. amidated gastrins in colon carcinogenesis has not been defined. We measured intermediate markers of carcinogenesis in transgenic mice overexpressing either progastrin (hGAS) or amidated gastrin (INS-GAS) in response to azoxymethane (AOM). The hGAS mice showed significantly higher numbers of aberrant crypt foci (140-200% increase) compared with that in wild-type (WT) and INS-GAS mice (P < 0.05) after AOM treatment. The bromodeoxyuridine-labeling index of colonic crypts also was significantly elevated in hGAS mice vs. that in WT and INS-GAS mice. The results therefore provide evidence for a mitogenic and cocarcinogenic role of nonamidated gastrins (progastrin), which is apparently not shared by the amidated gastrins. Although nonamidated gastrins are now believed to mediate mitogenic effects via novel receptors, amidated gastrins mediate biological effects via different receptor subtypes, which may explain the difference in the cocarcinogenic potential of nonamidated vs. amidated gastrins. In conclusion, our results provide strong support for a cocarcinogenic role for nonamidated gastrins in colon carcinogenesis.     

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.     

Gastrin biosynthesis in canine G cells

Gastrin requires extensive posttranslational processing for full biological activity. It is presumed that progastrin is cleaved at pairs of basic amino acids by a prohormone convertase to form a glycine-extended intermediate (G-Gly) that serves as a substrate for peptidyl-glycine alpha-amidating monooxygenase (PAM), resulting in COOH-terminally amidated gastrin. To confirm the nature of progastrin processing in a primary cell line, we performed [(35)S]methionine-labeled pulse-chase biosynthetic experiments in canine antral G cells. Radiolabeled progastrin reached a peak earlier than observed for G-Gly or amidated gastrin. G-Gly radioactivity accumulated in G cells and preceded the appearance of radioactivity in amidated gastrin. The conversion of G-Gly to amidated gastrin was enhanced by the PAM cofactor ascorbic acid. To determine whether one member of the prohormone convertase family (PC2) was responsible for progastrin cleavage, G cells were incubated with PC2 antisense oligonucleotide probes. Cells treated with antisense probes had reduced PC2 expression, an accumulation of radiolabeled progastrin, and a delay in the formation of amidated gastrin. Progastrin in antral G cells is cleaved via PC2 to form G-Gly that is converted to amidated gastrin via the actions of PAM.     

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.     

Achlorhydria induced changes in gastrin, somatostatin, H+/K(+)-ATPase and carbonic anhydrase in the sheep.

Gastrin, somatostatin, H+/K(+)-ATPase and carbonic anhydrase are principal elements of acid secretion. We investigated in the conscious sheep the effect of 24 h omeprazole (an H+/K(+)-ATPase inhibitor) infusion on these elements at the level of synthesis, storage and secretion. Omeprazole inhibited acid secretion-pH increased from 3.0 to 7.1 at 24 h. Plasma amidated and glycine extended gastrin increased 3-fold while the ratio of amidated to glycine extended gastrins (4:1) remained unchanged. Despite the increase in circulating gastrin, antral gastrin concentration and mRNA did not change significantly. Gastrin-17 (amidated and glycine extended) was the predominant form in the circulation and antrum, although there were preferential increases in larger forms following omeprazole treatment. Omeprazole had no effect on somatostatin mRNA or peptide levels in the fundus. Similarly, plasma somatostatin remained unchanged. However, antral somatostatin increased significantly (63%) following omeprazole treatment accompanied by a 4-fold increase in its mRNA. Fundic H+/K(+)-ATPase mRNA was unchanged but a significant increase (87%) in carbonic anhydrase II mRNA was observed. Omeprazole induced hypergastrinaemia occurred without a measurable reduction in storage or increased synthesis of gastrin at 24 h. Increased antral somatostatin synthesis and storage may result from stimulation by plasma gastrin on antral D cells, independent of acid. The rise in carbonic anhydrase II mRNA in the absence of any change in H+/K(+)-ATPase mRNA may reflect the differential sensitivity of the genes encoding these two enzymes to the stimulatory action of gastrin.     

Processing of the gastrin precursor. Modulation of phosphorylated, sulfated, and amidated products.

Post-translational processing of the precursor for rat gastrin yields products that include peptides phosphorylated at Ser96, amidated at Phe92, and sulfated at Tyr87 or Tyr103. The phosphorylation site is immediately adjacent to the processing point that gives rise to the biologically active amidated gastrins. We have examined changes in post-translational processing which occur in gastrin cells from rats that are physiologically stimulated (by feeding) or unstimulated (by fasting). Peptides were identified using site-directed radioimmunoassays and chromatographic systems that resolve phosphorylated, amidated, and sulfated progastrin products, including intermediates generated prior to amidation (i.e. C-terminal glycine-extended variants). Assays for Phe92-amidated peptides and for the C-terminal tryptic fragment of progastrin indicated decreases in the total tissue concentrations of immunoreactive peptide with fasting; in contrast, the tissue concentrations of glycine-extended biosynthetic intermediates were similar in fasted and fed rats. Taken together the data suggest a relative failure in amidation mechanisms in unstimulated cells. The endopeptidase cleavage of progastrin was not influenced significantly by fasting. However, the phosphorylation of peptide products containing Ser96 was depressed significantly in fasted rats. The proportions of amidated peptides sulfated at Tyr87 were generally lower than their corresponding glycine-extended biosynthetic precursors, but in both cases the proportion of peptide in the sulfated form was lower than for peptides sulfated at Tyr103. Feeding did not change the sulfation of amidated heptadecapeptide gastrin or its glycine-extended variant. The results suggest that the mechanisms determining phosphorylation and amidation of progastrin-related peptides depend on the patterns of stimulation of gastrin cells. The observation that decreased phosphorylation is associated with a failure to produce active amidated products is consistent with a regulatory function for phosphorylation in gastrin production.     

Progastrin maturation during ontogenesis. Accumulation of glycine-extended gastrins in rat antrum at weaning.

The post-translational maturation of antral progastrin was studied in the developing rat. While N-terminal proteolysis remained unchanged and tyrosine O-sulphation varied only slightly during ontogenesis, major changes were observed in the degree of alpha-carboxyamidation. In the third week of life the immediate precursor of amidated gastrin, glycine-extended gastrin, accumulated, and at weaning (day 21) the concentrations exceeded those of amidated gastrin. Our results confirm that weaning is accompanied by an increased synthesis of gastrin and imply that alpha-carboxyamidation is the rate-limiting step during the biosynthetic maturation of gastrin.     

The human gastrin precursor. Characterization of phosphorylated forms and fragments.

There is a potential phosphorylation site in the C-terminal region of the precursor for the acid-stimulating hormone gastrin, which is immediately adjacent to an important cleavage point. In the present study we have sought to identify, separate, quantify and characterize phosphorylated and unphosphorylated forms of human progastrin and its fragments. Identification was made by two radioimmunoassays: (a) a novel assay employing an antibody raised to intact human progastrin; and (b) an assay using antibody reacting with the C-terminal tryptic fragment of human progastrin, as well as progastrin itself. Two forms of human progastrin isolated from a gastrinoma were separated by ion-exchange h.p.l.c., and had similar elution positions on reverse-phase h.p.l.c. and on gel filtration. The more acidic peptide contained close to equimolar amounts of phosphate. On trypsinization, peptides were released that co-eluted on ion-exchange h.p.l.c. with, and had the immunochemical properties of, naturally occurring C-terminal fragments of progastrin. One of the latter was isolated and shown by Edman degradation after derivatization with ethanethiol to have the sequence Ser (P)-Ala-Glu-Asp-Glu-Asn. Similar peptides occur in antral mucosa resected from ulcer patients. The unphosphorylated forms of progastrin predominated, whereas the phosphorylated forms of the C-terminal fragments were predominant. This distribution could be explained by preferential cleavage of phosphorylated progastrin. We conclude that in human progastrin, Ser-96 can occur in the phosphorylated form; this residue immediately follows a pair of basic residues (Arg-Arg) that are cleaved during synthesis of the biologically active product.     

Plasma atrial natriuretic peptide and cyclic nucleotide levels before and after a marathon

Plasma alpha-atrial natriuretic peptide (alpha-ANP) concentration and levels of cyclic nucleotides [guanosine 3',5'-cyclic monophosphate (cGMP) and adenosine 3',5'-cyclic monophosphate (cAMP)] were studied in 23 runners before and after a marathon race. Blood samples were drawn from an antecubital vein the morning before the race (base line), at 3 P.M. (i.e., 2 h before the start), on arrival, and 12 and 36 h and 7 days later. Compared with the base-line values of plasma alpha-ANP (5 pmol/l), cGMP (3.8 nmol/l), and cAMP (15.8 nmol/l), the plasma levels of alpha-ANP, cGMP, and cAMP were increased immediately after the marathon, respectively, to 12.0 pmol/l, 12.7 nmol/l, and 50.5 nmol/l. The increase in the plasma alpha-ANP concentration was related (r = 0.85; P less than 0.001) to the changes in plasma cGMP, plasma lactate, hematocrit, and body weight. The plasma cGMP and cAMP concentrations had returned to the prerace levels 12 h after the marathon, whereas the plasma alpha-ANP concentration was significantly lower (3.1 pmol/l) than the base-line values and increased above the prerace values 36 h (7.5 pmol/l) and 7 days (6.8 pmol/l) after the marathon. The plasma cGMP level was also higher 36 h (5.4 nmol/l) and 7 days (5.0 nmol/l) after the marathon race.     

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.     

Identification and characterization of glycine-extended post-translational processing intermediates of progastrin in porcine stomach

We developed a radioimmunoassay specific for glycine-extended progastrin processing intermediates (G-Gly) using antisera generated against the synthetic peptide Tyr-Gly-Trp-Met-Asp-Phe-Gly. Distribution of immunoreactivity in the porcine gastrointestinal tract obtained with this antibody paralleled that of gastrin with the mucosa containing the highest quantity, 116 +/- 22 pmol/g, wet weight (mean +/- S.E., n = 5), or roughly 4% of gastrin concentration. This immunoreactivity was localized specifically to antral mucosal G-cells by immunohistochemistry. On Sephadex G-50 column chromatography of porcine antral mucosal extracts glycine-extended progastrin processing intermediates were separated into three principal molecular forms, each corresponding to known molecular forms of gastrin, component I, tetratriacontagastrin (G34) and heptadecagastrin (G17). Following purification by antibody-coupled affinity chromatography, one molecular form corresponding to G17 in size was shown to have an amino terminus identical to that of G17. Another molecular form corresponding to G34 in size could be converted to the molecular form corresponding to G17 by tryptic digestion. Our findings indicate that glycine-extended progastrin processing intermediates may serve as immediate precursors for each molecular form of gastrin, thus suggesting an alternative pathway for gastrin biosynthesis more complex than that previously conceived.     

Neurotensin-like immunoreactivities in human plasma: feeding responses and metabolism

Feeding responses and day and night levels of plasma concentration of neurotensin (NT) and NT-fragments were studied in healthy subjects. Plasma levels were measured by three radioimmunoassays recognizing intact NT in addition to C- and N-terminal immunoreactivity. The metabolism of NT was studied following intravenous administration. In 106 subjects fasting levels of intact NT (median 18 pmol/l), C-terminal (median 30 pmol/l) and N-terminal immunoreactivity (median 95 pmol/l) were unrelated to sex or age. Postprandially plasma levels in seven subjects measured with all assays increased by a factor 1-3. Following a mixed meal the increase was biphasic, whereas the response to dairy cream was monophasic. Repetitive measurements during 24 hours showed that levels of N-terminal immunoreactivity fluctuated in a manner related to meal ingestion and were elevated throughout the daytime, whereas intact NT and C-terminal immunoreactivity changed little. Following intravenous infusion of 2.4 pmol/kg/min NT in 5 subjects the chromatographic pattern was similar to that seen postprandially. The plasma half life of intact NT and C-terminal immunoreactivity was 1.5 and 1.2 min, whereas that of N-terminal immunoreactivity was 10.0 min. The differences in circulating levels could be explained by these differences in metabolism, but the physiological significance remains to be elucidated.     

Post-poly(Glu) cleavage and degradation modified by O-sulfated tyrosine: a novel post-translational processing mechanism.

Expression of bioactive peptides requires several modifications of the primary translation product. Gastrin, a vertebrate gut hormone, occurs in multiple forms, including a bioactive fragment of the predominant gastrin-17. Gastrin-17 is, however, without known cleavage sites. In order to identify the new site, we therefore isolated, from antral mucosa, fragments of gastrin-34 and -17 monitored by monospecific immunoassays. After three steps of reverse-phase chromatography, the short gastrins were identified as hepta-, hexa- and pentapeptide amides. By far the most abundant of these was tyrosine O-sulfated gastrin-6. The near complete sulfation contrasts with the larger gastrins, of which only half are sulfated. The longest N-terminal fragment of gastrin-34 was a hexadecapeptide without complementarity to the short gastrins. Instead, the predominant N-terminal fragment of gastrin-17 was the decapeptide complementary to gastrin-7. Therefore the novel processing site is the Glu10-Ala11 bond that follows a poly(Glu6-10) sequence. Moreover, gastrin-7 is apparently trimmed, with subsequent accumulation of sulfated gastrin-6. Consequently, O-sulfated tyrosine ensures production of a new hormone which stimulates gastric acid secretion as potently as gastrin-17.     

Biological activity and ferric ion binding of fragments of glycine-extended gastrin

Nonamidated gastrins such as progastrin and glycine-extended gastrin17 (Ggly) induce cell proliferation and migration in vitro and colonic mucosal proliferation in vivo. Our earlier NMR study defined the structure of Ggly and showed that ferric ions are essential to its biological activity, with the first binding to Glu7 and the second to Glu8 and Glu9 (Pannequin, J. et al. (2002) J. Biol. Chem. 277, 48602-48609). The aims of this study were to define the minimum biologically active fragment of Ggly and to determine whether ferric ions were also required for its activity. Cell-proliferation studies with Ggly fragments containing the five glutamate residues showed that the nonapeptide LE(5)AYG, the octapeptide LE(5)AY, and the heptapeptides E(5)AY and LE(5)A were fully active and that their activity was dependent on the presence of ferric ions. The activity of the hexapeptides LE(5) and E(5)A and the pentapeptide E(5) was reduced and independent of the presence of iron. The stoichiometry of ferric ion binding to LE(5)AYG, LE(5)AY, and E(5)AY, determined by absorption spectroscopy, was 2 mol/mol. NMR spectroscopy showed that the nonapeptide LE(5)AYG and shorter fragments had no defined structure and that the iron-binding sites differed from those in Ggly. We conclude that, in contrast to amidated gastrins where the C-terminal tetrapeptide is the minimum bioactive fragment, the shortest fully active fragments of Ggly are the heptapeptides LE(5)A and E(5)AY. These observations indicate that extensive proteolytic processing may not completely inactivate Ggly and that bioactive forms that are not detected by current radioimmunoassays may be present in tissues and/or plasma.     

Glucose dependent insulinotropic polypeptide (GIP) infused intravenously is insulinotropic in the fasting state in type 2 (non-insulin dependent) diabetes mellitus

The effects of an intravenous infusion of porcine GIP on beta-cell secretion in patients with untreated type 2 diabetes mellitus have been studied. The subjects were studied on two separate days. After a 10 h overnight fast and a further 120 min basal period they were given an intravenous infusion of porcine GIP (2 pmol.kg-1.min-1) or control solution in random order from 120-140 min. Frequent plasma glucose, insulin, C-peptide and GIP measurements were made throughout and the study was continued until 200 min. Plasma glucose levels were similar throughout both tests. During the GIP infusion there was an early significant rise in insulin concentration from 0.058 +/- 0.006 nmol/l to 0.106 +/- 0.007 nmol/l (P less than 0.01) within 6 min of commencing the GIP infusion and insulin levels reached a peak of 0.131 +/- 0.011 nmol/l at 10 min (P less than 0.01). Insulin levels remained significantly elevated during the rest of the GIP infusion (P less than 0.01-0.001) and returned to basal values 20 min post infusion. No change in basal insulin values was seen during the control infusion. C-peptide levels were similarly raised during the GIP infusion and the increase was significant just 4 min after commencing the GIP infusion (P less than 0.05). GIP levels increased from 16 +/- 3 pmol/l prior to the infusion to a peak of 286 +/- 24 pmol/l 20 min later. At 4 min when a significant beta-cell response was observed GIP levels were well within the physiological range.(ABSTRACT TRUNCATED AT 250 WORDS)     

Radioimmunoassay of atrial natriuretic peptide in human plasma: application to studies of volume and blood pressure homeostasis

Sensitive radioimmunoassay for determination of immunoreactive atrial natriuretic peptide (ANP) in human plasma was developed and employed for the study of plasma ANP concentrations in healthy controls under basal conditions (2.4 +/- 0.1 pmol/l) and during volume expansion by saline infusion (9.6 +/- 2.0 pmol/l and 14.2 +/- 1.8 pmol/l, respectively). Plasma renin activity and plasma aldosterone concentration exhibited opposite changes during saline infusion. In pathological states associated with extracellular fluid volume (ECFV) expansion, ANP concentration were significantly higher than in the controls (liver cirrhosis 8.6 +/- 0.9; congestive heart failure 33.1 +/- 4.8; chronic renal failure before haemodialysis 72.2 +/- 6.4 pmol/l). Further volume expansion in liver cirrhosis by saline infusion led to the further increase in ANP (13.3 +/- 1.3 and 16.1 +/- 1.5 pmol/l, respectively) and ECFV reduction by ultrafiltration during haemodialysis in chronic renal failure diminished but did not normalize plasma ANP (22.5 +/- 2.9 pmol/l). In patients with arterial hypertension the concentration of ANP exceeded the normal range by 62.5% and reached 8.0 +/- 0.5 pmol/l on the average. Our results support the suggestion that ANP is an important regulatory humoral mechanism participating in the regulation of sodium, volume and blood pressure homeostasis.     

PPARalpha agonists stimulate progastrin production in human colorectal carcinoma cells

The three subtypes of peroxisome proliferator activated-receptors (PPARalpha, delta and gamma) control the storage and metabolism of fatty acids. Treatment of rats with the PPARalpha ligand ciprofibrate increases serum gastrin concentrations, and several lines of evidence suggest that non-amidated gastrins act as growth factors for the colonic mucosa. The aim of the present study was to investigate the expression of PPARs and the effect of PPAR ligands on gastrin production and cell proliferation in human colorectal carcinoma (CRC) cell lines. mRNAs for all three PPAR subtypes were detected by PCR in all CRC cell lines tested. The concentrations of progastrin, but not of glycine-extended or amidated gastrin, measured by radioimmunoassay in LIM 1899 conditioned media and cell extracts were significantly increased by treatment with the PPARalpha ligand clofibrate. Similar increases in progastrin were seen following treatment with the PPARalpha ligands ciprofibrate and fenofibrate, but not with bezafibrate, gemfibrozil or Wy 14643. The PPARgamma agonist rosiglitazone had no significant effect on progastrin production. The PPARalpha ligand clofibrate also stimulated proliferation of the LIM 1899 cell line. We conclude that some PPARalpha ligands increase progastrin production by the human CRC cell line LIM 1899, and that clofibrate increases proliferation of LIM 1899 cells. These studies have revealed a relationship between PPARs and gastrin, two regulatory molecules implicated in the pathogenesis of CRC.     

Identification by specific radioimmunoassay of two novel peptides derived from the C-terminus of porcine preprogastrin

A radioimmunoassay has been developed using antibodies to a synthetic analogue of the C-terminal hexapeptide sequence of the porcine gastrin precursor. Boiling water extracts of porcine antral mucosa contained immunoreactive material that diluted in parallel with standard peptide. Concentrations of immunoreactivity were 5.5 +/- 0.8 nmol X g-1 (mean +/- S.E.M.) in antral mucosa and were closely similar to those of C-terminal heptadecapeptide gastrin immunoreactivity (5.0 +/- 0.6 nmol X g-1). Approximately 30-fold lower concentrations were found in porcine duodenum. A similar distribution was found in ferret, but human, rat and chicken antrum did not contain significant quantities of immunoreactivity. Gel filtration of porcine antral extracts on Sephadex G-50 revealed a single peak of immunoreactivity eluting in a similar position to G17, but on anion-exchange chromatography two peaks of immunoreactive material were separated. These also differed in their retention time on reverse phase HPLC. Both peptides are probably derived by tryptic cleavage at the C-terminus of porcine preprogastrin. No evidence was found to suggest that there are significant quantities of unprocessed preprogastrin in hog antral mucosa. The precise chemical difference between the two immunoreactive peptides identified here remains to be established; together, however, they provide specific markers for progastrin synthesis.