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Progastrin processing during antral G-cell hypersecretion in humans
GASTROENTEROLOGY
Progastrin Processing G-Cell Hypersecretion
1989;98:1083-70
During Antral in Humans
S. JENSEN, K. BORCH, L. HILSTED, and J. F. REHFELD University Department of Clinical Chemistry. Rigshospitalet, Copenhagen, Denmark; and Department of Surgery, LinkBping University Hospital, Sweden
Using radioimmunoassays specific for essential processing sites of human progastrin in comhination with chromatography before and after cleavage with trypsin and carhoxypeptidase B, we have examined antral biopsy specimens and serum from 10 hypergastrinemic patients with fundic atrophic gastritis and 7 normal control subjects. Four types of processing were studied: N-terminal proteolysis (at the N-terminus of component I, gastrin 34, and gastrin 17); C-terminal proteolysis (at the C-terminus of the amide donor, glycine,, in preprogastrin); cY-carhoxyamidation (of phenylalanine,,); and 0-sulfation (of tyrosine,,). The results show that progastrin during permanent G-cell hypersecretion is less completely processed with respect to Cterminal proteolysis, cy-amidation, and tyrosine-sulfation. In contrast, the degree of N-terminal proteolysis is normal. Thus, the processing of progastrin adjacent to the active site of gastrin is more restrictively controlled than N-terminal processing during G-cell hypersecretion associated with pernicious anemia. he gastrins (l-4) are synthesized by processing of the recently identified preprogastrin (5-7) at three dibasic and presumably one monobasic cleavage sites. Moreover, phenylalanine,, in preprogastrin is a-carboxyamidated and tyrosine,, partially O-sulfated (l-9, see also Figure 1). The different modifications govern both the bioactivity and immunoreactivity of the progastrin products (10-16).So far the interrelationship and completeness of the posttranslational modifications have been determined in normal antral tissue only (9). To understand and follow the pathogenetic effects of antral hypersecretion (IT-ZZ), however, it is also essential to know the processing of progastrin during chronic G-cell hypersecretion. Using radioimmunoassays specific for essential processing sites of human progastrin (15,23,24), we have now studied the processing in antral biopsy
T
specimens from patients with achlorhydria and antrum-sparing hypergastrinemic atrophic gastritis (25).The antral processing pattern was correlated to that of serum from the patients as well as to that of antral specimens and serum from normal control subjects.
Materials
and Methods
Serum and Antral Mucosal Specimens
Biopsy
After obtaining written informed consent, peripheral venous blood and gastroscopic antral biopsies were sampled from 10 fasting patients with hypergastrinemic atrophic gastritis associated with pernicious anemia and from 7 fasting normal subjects with normochlorhydria and normogastrinemia. The state of the fundic and antral mucosa was examined histologically in specimens from all patients and controls. The diagnostic criteria for pernicious anemia included the results of a Shilling and a pentagastrin test. Characteristics of patients and controls are shown in Table 1. The study was approved by the regional ethical committee.
Peptides A tetradecapeptide corresponding to sequence 517 of human gastrin 17 extended with glycine was synthesized by CRB Ltd. (Cambridge, U.K.), synthetic nonsulfated human gastrin 17 was obtained from Imperial Chemical Industries (Cheshire, U.K.), synthetic sulfated gastrin 17 from CRB Ltd., and nonsulfated gastrin 34 from Sigma [St. Louis, MO.). Sulfated gastrin 34 was a gift from R. A. Gregory [Liverpool, U.K.). We controlled purity and content of the peptides by reverse-phase high-performance liquid chromatography and amino acid analysis. The purity of the synthetic peptides was 295%.
0 1989 by the American Gastroenterological 0018-5085/89/$3.50
Association
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JENSEN ET AL.
GASTROENTEROLOGY Vol. 96. No.
ANTRAL PREPROGASTRIN
SIGNAL
ISPACER
1
21
ENDOPLASMATIC
PROCESSING
Arg-Arg
m ’
Gly-Arg-Arg
Lys-Lys
GASiRlN
40
4
57 58
34
’
74 75
101
9495
RETICULUM:
SIGNALASE
+
I TRANS-GOLGI
-___--TYROSYL-PROTEIN
APPARATUS
AND
SULFOTRANSFERASE
+
I
IMMATURE
SECRETORY
Figure
i
1.
VESICLES: TAYPSINLIKE
ENDOPEPTIDASE
t SECRETORY
GRANULES: TRYPSINLIKE
ENDOPEPTIDASES
+ 1.
$
Scheme of the normal posttranslational processing of human progastrin in antral G cells. The scheme is based partly on general knowledge about protein synthesis, partly on data from our laboratory (see references 6, 8, 9, and 15).
CARSOXYPEPTIDASE
+
+)-LIKE
ENZYME
I
TRYPSINLIKE
ENDOPEPTIDASES *
AMIDATING
a
ENZYME
s “‘I
2.
‘Gly -Gly
3
BIOACTIVE
7
GASTRINS
+
-
COMPONENT
CONH2
-
GASTRIN
34
CONH2
-
GASTRIN
17
(103 233), 750 heim, F.R.G.).
Enzymes treated
N-Tosyl-L-phenylalanine trypsin (LS 0003741),
chloromethyl 260 U/mg, was
ketonepurchased
Tissue
from Worthington Diagnostics Division (Freehold, N.J.); arylsulfatase (S-9754), 20-40 U/mg, from Sigma; and dicarboxypeptidase
isopropylfluorophosphate-treated Table
1. Characteristics Subjectsa
of Patients
With Hypergastrinemic
B
frozen. Atrophic
Age (yr) (median and
Sex
Group
n
range]
(F/Ml
Patients
10
69 (55-79)
614
Controls
7
62 (40-76)
314
n The gastrin concentrations
are given in picomoles
I
CONliz
J/ml, from Boehringer
Mannheim
(Mann-
Extractions
The antral biopsy specimens were immediately While frozen, the tissue was cut into pieces weighGastritis
per liter [median and 25%-75%
and Normogastrinemic
Control
Basal concentrations of amidated gastrin
Basal concentrations of glycine-extended
in serum (pmol/L)
gastrin in serum (pmol/L)
755 (440-1084) 914 ? 217 16 (2-32) 24 + 12 percentiles
(brackets)
106 (46-170) 109 + 21 5 (3-7) 7 + 1.7 as well as mean f SEMI.
April
1989
PROGASTRIN
ing a few milligrams, and immersed ml/g tissue) for 20 min, homogenized, 10,000 g for 30 min. The supernatant
Trypsin
and
B Cleavage
For determination of gastrin-Gly-Arg-Arg(-R) we used two types of sequential cleavage. The extracts (pH adjusted to 7.5) were incubated with equal volumes of 2 mg trypsiniml, 0.1 mol/L sodium phosphate buffer, pH 7.5, at 20°C for 30 min. The digestion was terminated by boiling for 10 min, each sample was centrifuged, and half of the supernatant was then incubated with carboxypeptidase B. The carboxypeptidase B cleavage was then performed by incubation in 0.1 mol/L sodium phosphate buffer, pH 7.5 (as above) with 100 ~1 carboxypeptidase Bi ml at 20°C for 30 min. This reaction was also terminated by boiling for 10 min. Tissue content of gastrin-Gly-Arg-Arg I-R) was calculated by comparing dilutions of the trypsinated extract to the trypsin and carboxypeptidase B digested extract. To evaluate the molecular size of the precursors, tissue extracts were chromatographed and the chromatographic fractions assayed before and after trypsin and carboxypeptidase B cleavage, as above.
The radioimmunoassays were performed as previ(15,23,24) using monoiodinated gastrin ously described (5-17) Gly tracer, nonsulfated gastrin (5-17) Gly standard, and antisera 3208 and 5284 for measurement of glycineextended gastrins. For measurement of amidated gastrins we used gastrin 17 tracer (26), nonsulfated gastrin 17 standard. and antisera 2604 and 2605. Antibody-bound and free tracer were separated using Amberlite resin CG4B.
Tissue extracts (n = 17) and hypergastrinemic sera (n = 10) were applied to long high-resolution columns (10 or 25 x 2000 mm) of Sephadex G-50 superfine, which were eluted with 0.125 mol/L NH,HCO,, pH 8.2, at 4°C with a Fractions of 1.2 flow rate of 4 and 18 ml/h, respectively. and 3.0 ml were collected. The columns were calibrated with ““I-albumin and ‘“NaCl for indication of void and total volume, respectively, and with sulfated and nonsulfated gastrin 34 and gastrin 17.
Statistical
Antisera
2. Distribution of Bioactive Amidated Extracts and Serum From Patients Material
Serum (hypergastrinemic extracts
patients] Antral extracts
755 (440-1084)
(hypergastrinemic (normal
Antral
amidated
Evaluated by component
Component (%I 21 (18-36)
pmol/L nmolig
Cl.0
22.1 + 3.6 nmolig 7.3 (3.9-18.5) nmol/g
a.0
12.7 k 5.3 nmolig ’ Values
are median
and 25-75s
percentiles
Products the percentage I, gastrin 34
[brackets]
I
Control
Subjects
distribution and gastrin
of 17
and in Antral 17
Gastrin 34 I%)
Gastrin (%I
57 (44-72)
22 (8-31)
6 (3-8)
94 (90-95)
2 217 pmoI/L
22.4 (10.7-32.5)
controls]
Progastrin
Gastrins in Antral Extracts From Normal With Hypergastrinemic Atrophic Gastritis”
914
Antral
Results
Total concentration patients)
Methods
Student’s t-test was used to assess the significance of the differences between the degrees of N-terminal proteolysis, tyrosineo-sulfation, phenylalanine a-carboxyamidation, and C-terminal proteolysis. Probability values ~0.05 were considered to be significant.
Two gastrin antisera were raised in rabbits against human carboxyamidated gastrin 17 (23). Antibody 2604 is C-terminal-specific and binds component I, gastrin 34, and gastrin 17 with equimolar potency irrespective of tyrosine(14). Antibody 2605 also recognizes the ami0-sulfation dated C-terminus of gastrin 17, but reacts poorly with sulfated gastrins, the cross-reactivity being 5% (14,24). Neither of the two antisera recognizes glycine-extended
Table
1065
Radioimmunoassay
Chromatography
Gastrin
IN HYPERGASTKINEMIA
gastrins. For measurement of glycine-extended gastrins we used antisera containing antibody 3208 and antibody 5284, which are both specific for glycine-extended gastrins. Their cross-reactivity with amidated gastrins is <0.2% (15). Both antibodies 3208 and 5284 bind glycineextended component I with reduced potency, antibody 5284 cross-reacts 60% and antibody 3208 50% (9). For the sake of clarity it should be remembered that component I is the largest bioactive (i.e., a-carboxyamidated) gastrin in serum and tissue (4,8,9,11). It is defined by the early elution by Sephadex G-50 chromatography (K,,. = 0.25) and by recognition of antisera specific for the C-terminal active site of the gastrins (4). The exact N-terminal sequence of component I remains to be determined, but Arg,, of human preprogastrin is a likely monobasic cleavage site (Figure 1). The precise sequence specificity, affinity, and binding homogeneity of all the antisera have been evaluated and described in detail elsewhere (14.15.23).
in boiling water (10 and centrifuged at was stored at -20°C.
Carboxypeptidase
PROCESSING
as well as mean
2 SEM.
6 ? 0.9 7 (4-10)
6.7 -t 1.5
92.5 + 1.8 93 (90-96)
93.6 t 1.6
1066
Table
JENSEN ET AL.
3.
GASTROENTEROLOGY
Distribution of Glycine-Extended Gastrins in AntraJ Extracts From Extracts and Serum From Patients With Hypergastrinemic Atrophic
Normal Control Gastritis”
Subjects
Vol. 96, No. 4
and in AntraJ
Glycine-extended I
Component Material Serum (hypergastrinemic
Total concentration patients]
pmol/L
106 (46-170)
Gastrin 34
(%I
Gastrin
(%I
58 (46-72)
42 (28-52)
17
(%I
109 2 21 pmol/L
Antral extracts
(hypergastrinemic
0.49 (0.36-0.87)
patients)
Antral extracts
(normal
0.05 (0.04-0.14) nmolig 0.10 2 0.03 nmol/g
controls]
u Values are median and 25%-75%
nmolig
+ 0.09 nmolig
0.60
percentiles
(brackets]
35 (31-13)
53 (47-65)
8.5 2 2.3
36.2 k 2.8
55.3
39 (34-42)
52 (44-60)
12 (3-14) 9.4 ?z 3.2
38.5
f
2.2
t 4.33
52.6 L 3.8
as well as mean ? SEM
Cl
A
10 (4-131
GASTRIN
C 17
GLYCINE-EXTENDED 15
15
10
10
GASTRIN
34
A
GLYCINE-EXTENDED
GASTRIN
I1 1
GASTRIN
17
34
5
5 COMPONENT
I
0
0 GLYCINE-EXTENDED GASTRIN
15
GASTRIN
34
15
17
,ne,
GLYCINE-EXTENDED 10
10 GASTRIN n*
34
GASTRIN
i I
5
5 COMPONENT
I
0
0
0
ELUTION
Figure
17
0.5
1.0
CONSTANT
of a neutral water extract of an antral mucosal biopsy specimen from a patient with hypergastrinemic 2. Gel-chromatography atrophic gastritis. One milliliter of extract was applied to a calibrated Sephadex G-50 superfine column (2.5 x 200 cm) eluted with 0.125 M NH,HCO, at a flow rate of 18 ml/h at 4’C. Fractions of 3.0 ml were collected and the concentrations of progastrin products measured with four different radioimmunoassay systems. Panel A shows the measurements using antiserum 2604 that binds a-carboxyamidated (-bioactive) gastrins with equimolar potency irrespective of N-terminal chain length and degree of tyrosine 0-sulfation. Panel B shows the measurements using antiserum 2605, which binds only nonsulfated. acarboxyamidated gastrins. Panel C shows the measurements using antiserum 3208. which binds glycine-extended gastrins. Finally, panel D shows the measurements using the same assay system as in panel C, but only after incubation of each fraction with trypsin and carboxypeptidase B. Thus, in addition to the glycine-extended progastrin fragments depicted in panel C, the measurements shown in panel D include progastrin(s) extended C-terminally beyond glycine.
April
PROGASTRIN
1989
PROCESSING
IN HYPEKCASTRINEMIA
1067
C
GLVCINE-EXTENDED GASTRIN 34 + GLVCINE-EXTENDED GASTRIN 17
COMPONENT
s
I
D GASTRIN ns
17 ‘PRO’-GASTRIN I 9
GLVCINE-EXTENDED GASTRIN 34 1
GLVCINE-EXTENDED GASTRIN 17
ELIJTION
CONSTANT (‘W
Figure
3. Gel-chromatography of a neutral water normogastrinemia. One milliliter of extract legend to Figure 2.
extract of an antral mucosal biopsy specimen from a control subject with was applied to a calibrated Sephadex G-50 superfine column as described in the
[Table 2) and the corresponding glycine-extended gastrins (Table 3), respectively, there were no differences in the degree of N-terminal proteolysis between the patients with hypergastrinemic atrophic gastritis and normal controls (p > 0.05; see also Figures 1, ZA, zC, 3A, and 3C). As shown by the reactivity with antiserum 3208 (specific for glycine-extended gastrins) after trypsin and carboxypeptidase B cleavage, the C-terminal dibasic cleavage site of progastrin is processed to a slightly lesser degree in the hypergastrinemic patients (Figures 1, 2D, and 30). The degree of 0-sulfation of tyrosine,, in the gastrins was significantly reduced in the hypersecretory antrum as judged by the reactivity with antiserum 2604 vs. antiserum 2605 (Table 4; p < 0.01). A similar reduction in the sulfated fractions of gastrin was apparent by gel chromatography (Figures ZA, 2B. 3A, and 3C).
Whereas antra from the control subjects contained only 0.7% glycine-extended gastrins in comparison with the amidated gastrins, antra from the hypergastrinemic patients contained more glycine-extended gastrins, i.e., 2.2% (Tables 2 and 3; p < 0.05).
Table
4.
Degree
of Tyrosine
0-Sulfation in Antral Extracts
Components
and Patients Gastritis”
of‘ the Gastrin From Controls
With Hypergastrinemic
Atrophic Sulfation
Group Hypergastrinemic Normal
patients
22 (15-26) 21.7 2 2.9 44 (31-46) 45.0 IfI 2.6
c.ontrols
” Values are median as mean + SEM.
ratio
WI
and Z5o/u-75%
percentiles
[brackets)
as well
1068
JENSEN
ET AL.
GASTROENTEROLOGY
A COMPONENT
I
GASTRIN t
t
1
1
‘)
Vol. 96, No. 4
C
34
GLVCINE-EXTENDED COMPONENT I !
GASTRIN ??ns
B
GL+VME;lNE;~~
17
GASTRIN-34 n. I
COMPONENT
0.5
0
1.0
ELUTION CONSTANT (Xav)
Figure
4. Gel-chromatography of serum from a patient with hypergastrinemic atrophic a Sephadex G-50 superfine column as described in the legend to Figure 2.
Circulating
Progastrin
Products
It was not possible to fractionate and characterize the progastrin products in serum from normal subjects due to the low concentrations (Table 1). Serum from the hypergastrinemic patients showed, however, a characteristic pattern (Tables 2 and 3, Figure 4) different from that of the antral extracts (Figures 2 and 3). Due to the difference in the metabolic clearance rates of large and small components, serum contained mainly large progastrin products such as the a-amidated component I and gastrin 34 (Figure 4A), the corresponding large glytine-extended gastrins (Figure 4C), and further Cterminally extended progastrins (Figure 4D). Notably, the low fraction of sulfated gastrins found in antral tissue from the hypergastrinemic patients (Table 4) was also observed in serum (Figures 4A and 4B).
Discussion The results of this study show that some of the posttranslational modifications of progastrin are less
gastritis.
One milliliter
of serum
was applied
to
complete in hypersecretory G ceils. Of the four types of modifications studied (Figure 1) only the Nterminal cleavages occurred in normal proportions (Figures 2 and 3). Thus, the high proportion of large molecular forms of gastrin in serum from the hypergastrinemic patients (Figure 4) reflects the slower clearance of long-chain gastrins rather than decreased N-terminal cleavage. Notably, the half-life of gastrin 34 is eight times longer than that of gastrin 17 (27,28), and that of component I is even longer (29). The decreased degree of tyrosine 0-sulfation confirms earlier results from our laboratory (30).Gastrin as such differs markedly from other sulfated polypeptides in being only partially sulfated (31,32). The explanation could be either that the amino acid residue immediately N-terminal to tyrosine in gastrin is neutral, whereas it is acidic in other peptides (32);or that the sulfotransferase in the G cells (Figure 1) is easily saturated, a phenomenon further exaggerated by increased synthesis (Table 4, Figures 2 and 4). It should be remembered, however, that although the degree of sulfation is decreased (Table 4), the number of sulfated gastrin molecules is increased in
April
1989
hypergastrinemic gastritis because of the grossly increased synthesis of gastrin., The most important processing sequence is Phe,,where trypsinlike cleavage Gly,,-Arg,,-Arg,,-Ser,,, of the Arg-Ser bond is followed by removal of arginine(s) by a carboxy-peptidase-B-like enzyme leaving glycine-extended gastrins as the immediate precursors of the bioactive gastrins (Figure 1). The crucial amidation process is then catalyzed by the copper-, ascorbic acid-, and oxygen-dependent amidation enzyme (33-35) in the secretory granules. In the present study these processings were less complete, as the percentages of glycine-extended and further C-terminal extended precursors were increased in hypergastrinemic atrophic gastritis. Even though the glycine-extended antral gastrins increased only from 0.7% in controls to 2.2% in the patients, the consequence is striking. Thus, the concentrations of glycine-extended gastrins in serum rose from 5 pmol/L in the controls to 106 pmol/L in patients with atrophic gastritis (Table 1). As the ratio between glycine-extended and amidated gastrin in antral veins and antral tissue is the same (36), the high proportion of glycine-extended gastrins in peripheral serum indicates that glycine-extended component I and gastrin 34 (Figure 4C) are cleared at a slower rate than the corresponding amidated forms. Similar mechanisms may explain the high concentrations of further C-terminal extended gastrins in peripheral serum (Figure 4D), whereas constitutive secretion (37) from the G cells has not been reported so far. In addition to the modifications examined in this study, preprogastrin undergoes two further modifications. First, the N-terminal signal peptide is removed cotranslationally in the rough endoplasmatic reticulum (Figure 1). Our approach does not allow estimates of this signalase activity. Second, serine,, phosphorylation has recently been described (38). Such modification might influence the processing of the amidation site, and consequently play a role in the processing observed in this study. The combination of decreased processing and slow clearance changes the plasma profile markedly. Thus, progastrin normally matures almost completely to bioactive, amidated gastrins, which constitute the predominant products of progastrin in normal plasma (39,40). The changed profile in hypergastrinemic atrophic gastritis therefore has two implications. First, accurate measurements of antral G-cell activity cannot be based on assays that measure only amidated gastrins in plasma. Second, as antral hypersecretion is accompanied for example, by enterochromaffinlike-cell proliferation (18,20-22) and disturbed islet cell growth (17) and secretion (19), it becomes important to examine whether non-
PROGASTRIN
PROCESSING
IN HYPERGASTRINEMIA
1069
amidated progastrin products in plasma may influence these effects or whether they are due solely to cY-carboxyamidated gastrins. Two recent studies have also examined human antral progastrin (41,42).Unfortunately, they are difficult to compare with the present study, because both material and methodology differ considerably. Nevertheless, Del Valle et al. (42)found antral glytine-extended gastrins in concentrations similar to those in our controls (Table 3). The two studies also examined hypersecretory tissue, namely gastrinoma extracts (41,42).The mechanisms behind the increased gastrin synthesis in pancreatic tumor cells and in achlorhydric antral G cells make the results difficult to compare. The only common feature seems to be that the concentration of progastrin generally is increased in tissue with enhanced synthesis. References 1. Gregory RA, Tracy HJ. The constitution and properties of two gastrins extracted from hog antral mucosa. Gut 1964;5:10317. of two “big gastrins” from 2. Gregory RA. Tracy HJ. Isolation Zollinger-Ellison tumour tissue. Lancet 1972;ii:797-9. of two mini-gastrins from 3. Gregory RA. Tracy HJ. Isolation Zollinger-Ellison tumour tissue. Gut 1974;15:683-6. 4. Rehfeld JF. Three components of gastrin in human serum. Biochim Biophys Acta 1972;285:364-72. 5. Yoo OJ, Powell CT, Agarwal K. Molecular cloning and nucleotide sequence of full-length cDNA coding for porcine gastrin. Proc Nat1 Acad Sci USA 1982;79:1049-53. cloning of human 6. Boel E, Vuust J. Norris K. et al. Molecular gastrin cDNA: evidence for evolution of gastrin by gene duplication, Proc Nat1 Acad Sci USA 1983:80:2866-9. 7. Fuller PJ. Stone D, Brand SJ. Molecular cloning and sequencing of a rat preprogastrin complementary deoxyribonucleic acid. Mol Endocrinol 1987;1:306-11. 8. Brand SJ, Klarlund J, Schwartz TW, Rehfeld JF. Biosynthesis of tyrosine O-sulfated gastrins in rat antral mucosa. J Biol Chem 1984:259:13246-52. 9. Hilsted L. Rehfeld JF. Alpha-carboxpamidation of antral progastrin: relation to other post-translational modifications. J Biol Chem 1987;262:16953-7. 10. Morley JS. Tracy HJ, Gregory RA. Structure-function relationships in the active C-terminal tetrapeptide sequence of gastrin. Nature 1965;207:1356-9. 11 Jensen SL. Rehfeld JF, Holst JJ, Fahrenkrug J. Nielsen OV, Schaffalitzky de Muckadell OB. Secretory effects of the gastrins on the isolated perfused porcine pancreas. Am J Physiol 1980;238:E186-92. 12 Hilsted L, Hint K. Christiansen J. Rehfeld JF. Neither glycineextended gastrin nor the l-13 fragment of gastrin-17 influences gastric acid secretion in humans. Gastroenterology 1988;94:96-102. 13 De Magistris L. Rehfeld JF. A simple enzymatic procedure for radioimmunochemical quantitation of the large molecular forms of gastrin and cholecystokinin Anal Biochem 1980; 102:126-33. 14. Rehfeld JF, de Magistris L, Andersen BN. Sulfation of gastrin: effect on immunoreactivity. Regul Pept 1981:2:333-42.
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15. Hilsted L, Rehfeld JF. Measurement of precursors for aamidated hormones by radioimmunoassay of glycine-extended peptides after trypsin-carboxypeptidase B cleavage. Anal Biochem 1986;152:119-26. 16. Dockray GJ, Walsh JH. Aminoterminal gastrin fragment in serum of Zollinger-Ellison syndrome patients. Gastroenterology 1975;68:222-30. 17. Larsson LI, Ljungberg 0, Sundler F, et al. Antropyloric gastrinoma associated with pancreatic nesidioblastosis and proliferation of islets. Virchows Arch 1973;360:305-14. 18. Borch K, Renvall H, Liedberg G, Andersen BN. Relations between circulating gastrin and endocrine cell proliferation in the atrophic gastric fundic mucosa. Stand J Gastroenterol 1986;21:357-63. 19. Rehfeld JF. Disturbed islet-cell function related to endogenous gastrin release: studies on insulin secretion and glucose tolerance in pernicious anemia. J Clin Invest 1976;58:41-9. 20. Larsson LI, Rehfeld JF, Stockbrugger R, et al. Mixed endocrine gastric tumors associated with hypergastrinemia of antral origin. Am J Path01 1978;93:53-68. 21. Alumets J, El Munshid HA, Hlkanson R, et al. Effect of antrum exclusion on endocrine cells of rat stomach. J Physiol 1979;286:145-55. 22. Borch K, Renvall H, Liedberg G. Gastric endocrine cell hyperplasia and carcinoid tumors in pernicious anemia. Gastroenterology 1985;88:638-48. 23. Rehfeld JF, Stadil F, Rubin B. Production and evaluation of antibodies for the radioimmunoassay of gastrin. Stand J Clin Lab Invest 1972;30:221-32. 24. Andersen BN, de Magistris L, Rehfeld JF. Radioimmunochemical quantitation of sulfated and nonsulfated gastrins in serum. Clin Chim Acta 1983;127:29-39. 25. Borch K, Renvall H, Kullman E, Wilander E. Gastric carcinoid associated with the syndrome of hypergastrinemic atrophic gastritis. A prospective analysis of 11 cases. Am J Surg Path01 1987;11:435-44. 26. Stadil F, Rehfeld JF. Preparation and evaluation of “‘Isynthetic human gastrin-17 for radioimmunoanalysis. Stand J Clin Lab Invest 1972;30:361-9. 27. Walsh JH, Debas HT, Grossman MI. Pure human big gastrin: immunochemical properties, disappearance half-time, and acid-stimulating action in dogs. J Clin Invest 1974;54:477-85. 28. Walsh JH, Isenberg JI. Ansfield J, Maxwell V. Clearance and acid-stimulating action of human big and little gastrins in duodenal ulcer subjects. J Clin Invest 1976;57:1125-31. 29. Fahrenkrug J, Schaffalitzky de Muckadell OB, Hornum I, Rehfeld JF. The mechanism of hypergastrinemia in achlorhydria: effect of food, acid and calcitonin on serum gastrin concentrations and component pattern in pernicious anemia. Gastroenterology 1976;71:33-8. 30. Andersen BN, Petersen B, Borch K. Decreased sulfation of
GASTROENTEROLOGY
31. 32. 33.
34.
35.
36.
37. 38.
39. 40.
41.
42.
Vol. 96, No. 4
serum and tissue gastrin in hypergastrinemia of antral origin. Digestion 1985;31:17-24. Andersen BN. Species variation in the tyrosine sulfation of mammalian gastrins. Gen Comp Endocrinol 1985;58:44-50. Huttner WB, Baeuerle PA. Proteinsulfation of tyrosine. Modern Cell Biol 1988;6 (in press). Bradbury AF, Finnie MDA, Smyth DG. Mechanism of Cterminal amide formation by pituitary enzymes. Nature 1982; 298:686-8. Murthy ASN, Mains RE, Eipper BA. Purification and characterization of peptidylglycine a-amidating monooxygenase from bovine neurointermediate pituitary. J Biol Chem 1986; 261:1815-22. Park LP. Thiele EA, Dickerson IM, Mains RE, Eipper BA. Cloning of cDNA encoding bovine peptidyl-glycine a-amidating monooxygenase. In: Christiansen C, Riis BJ, eds. Highlights on endocrinology (Proceedings of the First European Congress on Endocrinology) Copenhagen: Ndrhaven Print, 1987:133-40. Hilsted L, Hansen CP. Co-release of amidated and glycineextended antral gastrins after a meal. Am J Physiol 1988 255;G665-9. Kelly RB. Pathways of protein secretion in eucaryotes. Science 1985;230:25-32. Dockray GJ, Varro A, Desmond H, Young J. Gregory H, Gregory RA. Post-translational processing of the porcine gastrin precursor by phosphorylation of the COOH-terminal fragment. J Biol Chem 1987;262:8643-7. Gregory RA. A review of some recent developments in the chemistry of the gastrins. Bioorg Khim 1979;8:497-511. Taylor IL, Dockray GJ. Calam J, Walker R. Big and little gastrin responses to food in normal and ulcer subjects. Gut 1979;20: 957-62. Pauwels S, Desmond H, Dimaline R, Dockray GJ. Identification of progastrin in gastrinomas, antrum and duodenum by a novel radioimmunoassay. J Clin Invest 1986;77:37681. Del Valle J, Sugano K. Yamada T. Progastrin and its glycineextended posttranslational processing intermediates in human gastrointestinal tissues. Gastroenterology 1987;92:190812.
Received March 15, 1988. Accepted November 10, 1988. Address requests for reprints to: Jens F. Rehfeld, M.D.. Department of Clinical Chemistry. Rigshospitalet, DK-2100, Copenhagen, Denmark. This study was supported by grants from the Danish Medical Research Council, and the Lundbeck and Willumsen Foundations. The authors thank Aase Valsted and Lisbeth Andreasen for skillful technical assistance.
Progastrin Processing G-Cell Hypersecretion
1989;98:1083-70
During Antral in Humans
S. JENSEN, K. BORCH, L. HILSTED, and J. F. REHFELD University Department of Clinical Chemistry. Rigshospitalet, Copenhagen, Denmark; and Department of Surgery, LinkBping University Hospital, Sweden
Using radioimmunoassays specific for essential processing sites of human progastrin in comhination with chromatography before and after cleavage with trypsin and carhoxypeptidase B, we have examined antral biopsy specimens and serum from 10 hypergastrinemic patients with fundic atrophic gastritis and 7 normal control subjects. Four types of processing were studied: N-terminal proteolysis (at the N-terminus of component I, gastrin 34, and gastrin 17); C-terminal proteolysis (at the C-terminus of the amide donor, glycine,, in preprogastrin); cY-carhoxyamidation (of phenylalanine,,); and 0-sulfation (of tyrosine,,). The results show that progastrin during permanent G-cell hypersecretion is less completely processed with respect to Cterminal proteolysis, cy-amidation, and tyrosine-sulfation. In contrast, the degree of N-terminal proteolysis is normal. Thus, the processing of progastrin adjacent to the active site of gastrin is more restrictively controlled than N-terminal processing during G-cell hypersecretion associated with pernicious anemia. he gastrins (l-4) are synthesized by processing of the recently identified preprogastrin (5-7) at three dibasic and presumably one monobasic cleavage sites. Moreover, phenylalanine,, in preprogastrin is a-carboxyamidated and tyrosine,, partially O-sulfated (l-9, see also Figure 1). The different modifications govern both the bioactivity and immunoreactivity of the progastrin products (10-16).So far the interrelationship and completeness of the posttranslational modifications have been determined in normal antral tissue only (9). To understand and follow the pathogenetic effects of antral hypersecretion (IT-ZZ), however, it is also essential to know the processing of progastrin during chronic G-cell hypersecretion. Using radioimmunoassays specific for essential processing sites of human progastrin (15,23,24), we have now studied the processing in antral biopsy
T
specimens from patients with achlorhydria and antrum-sparing hypergastrinemic atrophic gastritis (25).The antral processing pattern was correlated to that of serum from the patients as well as to that of antral specimens and serum from normal control subjects.
Materials
and Methods
Serum and Antral Mucosal Specimens
Biopsy
After obtaining written informed consent, peripheral venous blood and gastroscopic antral biopsies were sampled from 10 fasting patients with hypergastrinemic atrophic gastritis associated with pernicious anemia and from 7 fasting normal subjects with normochlorhydria and normogastrinemia. The state of the fundic and antral mucosa was examined histologically in specimens from all patients and controls. The diagnostic criteria for pernicious anemia included the results of a Shilling and a pentagastrin test. Characteristics of patients and controls are shown in Table 1. The study was approved by the regional ethical committee.
Peptides A tetradecapeptide corresponding to sequence 517 of human gastrin 17 extended with glycine was synthesized by CRB Ltd. (Cambridge, U.K.), synthetic nonsulfated human gastrin 17 was obtained from Imperial Chemical Industries (Cheshire, U.K.), synthetic sulfated gastrin 17 from CRB Ltd., and nonsulfated gastrin 34 from Sigma [St. Louis, MO.). Sulfated gastrin 34 was a gift from R. A. Gregory [Liverpool, U.K.). We controlled purity and content of the peptides by reverse-phase high-performance liquid chromatography and amino acid analysis. The purity of the synthetic peptides was 295%.
0 1989 by the American Gastroenterological 0018-5085/89/$3.50
Association
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JENSEN ET AL.
GASTROENTEROLOGY Vol. 96. No.
ANTRAL PREPROGASTRIN
SIGNAL
ISPACER
1
21
ENDOPLASMATIC
PROCESSING
Arg-Arg
m ’
Gly-Arg-Arg
Lys-Lys
GASiRlN
40
4
57 58
34
’
74 75
101
9495
RETICULUM:
SIGNALASE
+
I TRANS-GOLGI
-___--TYROSYL-PROTEIN
APPARATUS
AND
SULFOTRANSFERASE
+
I
IMMATURE
SECRETORY
Figure
i
1.
VESICLES: TAYPSINLIKE
ENDOPEPTIDASE
t SECRETORY
GRANULES: TRYPSINLIKE
ENDOPEPTIDASES
+ 1.
$
Scheme of the normal posttranslational processing of human progastrin in antral G cells. The scheme is based partly on general knowledge about protein synthesis, partly on data from our laboratory (see references 6, 8, 9, and 15).
CARSOXYPEPTIDASE
+
+)-LIKE
ENZYME
I
TRYPSINLIKE
ENDOPEPTIDASES *
AMIDATING
a
ENZYME
s “‘I
2.
‘Gly -Gly
3
BIOACTIVE
7
GASTRINS
+
-
COMPONENT
CONH2
-
GASTRIN
34
CONH2
-
GASTRIN
17
(103 233), 750 heim, F.R.G.).
Enzymes treated
N-Tosyl-L-phenylalanine trypsin (LS 0003741),
chloromethyl 260 U/mg, was
ketonepurchased
Tissue
from Worthington Diagnostics Division (Freehold, N.J.); arylsulfatase (S-9754), 20-40 U/mg, from Sigma; and dicarboxypeptidase
isopropylfluorophosphate-treated Table
1. Characteristics Subjectsa
of Patients
With Hypergastrinemic
B
frozen. Atrophic
Age (yr) (median and
Sex
Group
n
range]
(F/Ml
Patients
10
69 (55-79)
614
Controls
7
62 (40-76)
314
n The gastrin concentrations
are given in picomoles
I
CONliz
J/ml, from Boehringer
Mannheim
(Mann-
Extractions
The antral biopsy specimens were immediately While frozen, the tissue was cut into pieces weighGastritis
per liter [median and 25%-75%
and Normogastrinemic
Control
Basal concentrations of amidated gastrin
Basal concentrations of glycine-extended
in serum (pmol/L)
gastrin in serum (pmol/L)
755 (440-1084) 914 ? 217 16 (2-32) 24 + 12 percentiles
(brackets)
106 (46-170) 109 + 21 5 (3-7) 7 + 1.7 as well as mean f SEMI.
April
1989
PROGASTRIN
ing a few milligrams, and immersed ml/g tissue) for 20 min, homogenized, 10,000 g for 30 min. The supernatant
Trypsin
and
B Cleavage
For determination of gastrin-Gly-Arg-Arg(-R) we used two types of sequential cleavage. The extracts (pH adjusted to 7.5) were incubated with equal volumes of 2 mg trypsiniml, 0.1 mol/L sodium phosphate buffer, pH 7.5, at 20°C for 30 min. The digestion was terminated by boiling for 10 min, each sample was centrifuged, and half of the supernatant was then incubated with carboxypeptidase B. The carboxypeptidase B cleavage was then performed by incubation in 0.1 mol/L sodium phosphate buffer, pH 7.5 (as above) with 100 ~1 carboxypeptidase Bi ml at 20°C for 30 min. This reaction was also terminated by boiling for 10 min. Tissue content of gastrin-Gly-Arg-Arg I-R) was calculated by comparing dilutions of the trypsinated extract to the trypsin and carboxypeptidase B digested extract. To evaluate the molecular size of the precursors, tissue extracts were chromatographed and the chromatographic fractions assayed before and after trypsin and carboxypeptidase B cleavage, as above.
The radioimmunoassays were performed as previ(15,23,24) using monoiodinated gastrin ously described (5-17) Gly tracer, nonsulfated gastrin (5-17) Gly standard, and antisera 3208 and 5284 for measurement of glycineextended gastrins. For measurement of amidated gastrins we used gastrin 17 tracer (26), nonsulfated gastrin 17 standard. and antisera 2604 and 2605. Antibody-bound and free tracer were separated using Amberlite resin CG4B.
Tissue extracts (n = 17) and hypergastrinemic sera (n = 10) were applied to long high-resolution columns (10 or 25 x 2000 mm) of Sephadex G-50 superfine, which were eluted with 0.125 mol/L NH,HCO,, pH 8.2, at 4°C with a Fractions of 1.2 flow rate of 4 and 18 ml/h, respectively. and 3.0 ml were collected. The columns were calibrated with ““I-albumin and ‘“NaCl for indication of void and total volume, respectively, and with sulfated and nonsulfated gastrin 34 and gastrin 17.
Statistical
Antisera
2. Distribution of Bioactive Amidated Extracts and Serum From Patients Material
Serum (hypergastrinemic extracts
patients] Antral extracts
755 (440-1084)
(hypergastrinemic (normal
Antral
amidated
Evaluated by component
Component (%I 21 (18-36)
pmol/L nmolig
Cl.0
22.1 + 3.6 nmolig 7.3 (3.9-18.5) nmol/g
a.0
12.7 k 5.3 nmolig ’ Values
are median
and 25-75s
percentiles
Products the percentage I, gastrin 34
[brackets]
I
Control
Subjects
distribution and gastrin
of 17
and in Antral 17
Gastrin 34 I%)
Gastrin (%I
57 (44-72)
22 (8-31)
6 (3-8)
94 (90-95)
2 217 pmoI/L
22.4 (10.7-32.5)
controls]
Progastrin
Gastrins in Antral Extracts From Normal With Hypergastrinemic Atrophic Gastritis”
914
Antral
Results
Total concentration patients)
Methods
Student’s t-test was used to assess the significance of the differences between the degrees of N-terminal proteolysis, tyrosineo-sulfation, phenylalanine a-carboxyamidation, and C-terminal proteolysis. Probability values ~0.05 were considered to be significant.
Two gastrin antisera were raised in rabbits against human carboxyamidated gastrin 17 (23). Antibody 2604 is C-terminal-specific and binds component I, gastrin 34, and gastrin 17 with equimolar potency irrespective of tyrosine(14). Antibody 2605 also recognizes the ami0-sulfation dated C-terminus of gastrin 17, but reacts poorly with sulfated gastrins, the cross-reactivity being 5% (14,24). Neither of the two antisera recognizes glycine-extended
Table
1065
Radioimmunoassay
Chromatography
Gastrin
IN HYPERGASTKINEMIA
gastrins. For measurement of glycine-extended gastrins we used antisera containing antibody 3208 and antibody 5284, which are both specific for glycine-extended gastrins. Their cross-reactivity with amidated gastrins is <0.2% (15). Both antibodies 3208 and 5284 bind glycineextended component I with reduced potency, antibody 5284 cross-reacts 60% and antibody 3208 50% (9). For the sake of clarity it should be remembered that component I is the largest bioactive (i.e., a-carboxyamidated) gastrin in serum and tissue (4,8,9,11). It is defined by the early elution by Sephadex G-50 chromatography (K,,. = 0.25) and by recognition of antisera specific for the C-terminal active site of the gastrins (4). The exact N-terminal sequence of component I remains to be determined, but Arg,, of human preprogastrin is a likely monobasic cleavage site (Figure 1). The precise sequence specificity, affinity, and binding homogeneity of all the antisera have been evaluated and described in detail elsewhere (14.15.23).
in boiling water (10 and centrifuged at was stored at -20°C.
Carboxypeptidase
PROCESSING
as well as mean
2 SEM.
6 ? 0.9 7 (4-10)
6.7 -t 1.5
92.5 + 1.8 93 (90-96)
93.6 t 1.6
1066
Table
JENSEN ET AL.
3.
GASTROENTEROLOGY
Distribution of Glycine-Extended Gastrins in AntraJ Extracts From Extracts and Serum From Patients With Hypergastrinemic Atrophic
Normal Control Gastritis”
Subjects
Vol. 96, No. 4
and in AntraJ
Glycine-extended I
Component Material Serum (hypergastrinemic
Total concentration patients]
pmol/L
106 (46-170)
Gastrin 34
(%I
Gastrin
(%I
58 (46-72)
42 (28-52)
17
(%I
109 2 21 pmol/L
Antral extracts
(hypergastrinemic
0.49 (0.36-0.87)
patients)
Antral extracts
(normal
0.05 (0.04-0.14) nmolig 0.10 2 0.03 nmol/g
controls]
u Values are median and 25%-75%
nmolig
+ 0.09 nmolig
0.60
percentiles
(brackets]
35 (31-13)
53 (47-65)
8.5 2 2.3
36.2 k 2.8
55.3
39 (34-42)
52 (44-60)
12 (3-14) 9.4 ?z 3.2
38.5
f
2.2
t 4.33
52.6 L 3.8
as well as mean ? SEM
Cl
A
10 (4-131
GASTRIN
C 17
GLYCINE-EXTENDED 15
15
10
10
GASTRIN
34
A
GLYCINE-EXTENDED
GASTRIN
I1 1
GASTRIN
17
34
5
5 COMPONENT
I
0
0 GLYCINE-EXTENDED GASTRIN
15
GASTRIN
34
15
17
,ne,
GLYCINE-EXTENDED 10
10 GASTRIN n*
34
GASTRIN
i I
5
5 COMPONENT
I
0
0
0
ELUTION
Figure
17
0.5
1.0
CONSTANT
of a neutral water extract of an antral mucosal biopsy specimen from a patient with hypergastrinemic 2. Gel-chromatography atrophic gastritis. One milliliter of extract was applied to a calibrated Sephadex G-50 superfine column (2.5 x 200 cm) eluted with 0.125 M NH,HCO, at a flow rate of 18 ml/h at 4’C. Fractions of 3.0 ml were collected and the concentrations of progastrin products measured with four different radioimmunoassay systems. Panel A shows the measurements using antiserum 2604 that binds a-carboxyamidated (-bioactive) gastrins with equimolar potency irrespective of N-terminal chain length and degree of tyrosine 0-sulfation. Panel B shows the measurements using antiserum 2605, which binds only nonsulfated. acarboxyamidated gastrins. Panel C shows the measurements using antiserum 3208. which binds glycine-extended gastrins. Finally, panel D shows the measurements using the same assay system as in panel C, but only after incubation of each fraction with trypsin and carboxypeptidase B. Thus, in addition to the glycine-extended progastrin fragments depicted in panel C, the measurements shown in panel D include progastrin(s) extended C-terminally beyond glycine.
April
PROGASTRIN
1989
PROCESSING
IN HYPEKCASTRINEMIA
1067
C
GLVCINE-EXTENDED GASTRIN 34 + GLVCINE-EXTENDED GASTRIN 17
COMPONENT
s
I
D GASTRIN ns
17 ‘PRO’-GASTRIN I 9
GLVCINE-EXTENDED GASTRIN 34 1
GLVCINE-EXTENDED GASTRIN 17
ELIJTION
CONSTANT (‘W
Figure
3. Gel-chromatography of a neutral water normogastrinemia. One milliliter of extract legend to Figure 2.
extract of an antral mucosal biopsy specimen from a control subject with was applied to a calibrated Sephadex G-50 superfine column as described in the
[Table 2) and the corresponding glycine-extended gastrins (Table 3), respectively, there were no differences in the degree of N-terminal proteolysis between the patients with hypergastrinemic atrophic gastritis and normal controls (p > 0.05; see also Figures 1, ZA, zC, 3A, and 3C). As shown by the reactivity with antiserum 3208 (specific for glycine-extended gastrins) after trypsin and carboxypeptidase B cleavage, the C-terminal dibasic cleavage site of progastrin is processed to a slightly lesser degree in the hypergastrinemic patients (Figures 1, 2D, and 30). The degree of 0-sulfation of tyrosine,, in the gastrins was significantly reduced in the hypersecretory antrum as judged by the reactivity with antiserum 2604 vs. antiserum 2605 (Table 4; p < 0.01). A similar reduction in the sulfated fractions of gastrin was apparent by gel chromatography (Figures ZA, 2B. 3A, and 3C).
Whereas antra from the control subjects contained only 0.7% glycine-extended gastrins in comparison with the amidated gastrins, antra from the hypergastrinemic patients contained more glycine-extended gastrins, i.e., 2.2% (Tables 2 and 3; p < 0.05).
Table
4.
Degree
of Tyrosine
0-Sulfation in Antral Extracts
Components
and Patients Gastritis”
of‘ the Gastrin From Controls
With Hypergastrinemic
Atrophic Sulfation
Group Hypergastrinemic Normal
patients
22 (15-26) 21.7 2 2.9 44 (31-46) 45.0 IfI 2.6
c.ontrols
” Values are median as mean + SEM.
ratio
WI
and Z5o/u-75%
percentiles
[brackets)
as well
1068
JENSEN
ET AL.
GASTROENTEROLOGY
A COMPONENT
I
GASTRIN t
t
1
1
‘)
Vol. 96, No. 4
C
34
GLVCINE-EXTENDED COMPONENT I !
GASTRIN ??ns
B
GL+VME;lNE;~~
17
GASTRIN-34 n. I
COMPONENT
0.5
0
1.0
ELUTION CONSTANT (Xav)
Figure
4. Gel-chromatography of serum from a patient with hypergastrinemic atrophic a Sephadex G-50 superfine column as described in the legend to Figure 2.
Circulating
Progastrin
Products
It was not possible to fractionate and characterize the progastrin products in serum from normal subjects due to the low concentrations (Table 1). Serum from the hypergastrinemic patients showed, however, a characteristic pattern (Tables 2 and 3, Figure 4) different from that of the antral extracts (Figures 2 and 3). Due to the difference in the metabolic clearance rates of large and small components, serum contained mainly large progastrin products such as the a-amidated component I and gastrin 34 (Figure 4A), the corresponding large glytine-extended gastrins (Figure 4C), and further Cterminally extended progastrins (Figure 4D). Notably, the low fraction of sulfated gastrins found in antral tissue from the hypergastrinemic patients (Table 4) was also observed in serum (Figures 4A and 4B).
Discussion The results of this study show that some of the posttranslational modifications of progastrin are less
gastritis.
One milliliter
of serum
was applied
to
complete in hypersecretory G ceils. Of the four types of modifications studied (Figure 1) only the Nterminal cleavages occurred in normal proportions (Figures 2 and 3). Thus, the high proportion of large molecular forms of gastrin in serum from the hypergastrinemic patients (Figure 4) reflects the slower clearance of long-chain gastrins rather than decreased N-terminal cleavage. Notably, the half-life of gastrin 34 is eight times longer than that of gastrin 17 (27,28), and that of component I is even longer (29). The decreased degree of tyrosine 0-sulfation confirms earlier results from our laboratory (30).Gastrin as such differs markedly from other sulfated polypeptides in being only partially sulfated (31,32). The explanation could be either that the amino acid residue immediately N-terminal to tyrosine in gastrin is neutral, whereas it is acidic in other peptides (32);or that the sulfotransferase in the G cells (Figure 1) is easily saturated, a phenomenon further exaggerated by increased synthesis (Table 4, Figures 2 and 4). It should be remembered, however, that although the degree of sulfation is decreased (Table 4), the number of sulfated gastrin molecules is increased in
April
1989
hypergastrinemic gastritis because of the grossly increased synthesis of gastrin., The most important processing sequence is Phe,,where trypsinlike cleavage Gly,,-Arg,,-Arg,,-Ser,,, of the Arg-Ser bond is followed by removal of arginine(s) by a carboxy-peptidase-B-like enzyme leaving glycine-extended gastrins as the immediate precursors of the bioactive gastrins (Figure 1). The crucial amidation process is then catalyzed by the copper-, ascorbic acid-, and oxygen-dependent amidation enzyme (33-35) in the secretory granules. In the present study these processings were less complete, as the percentages of glycine-extended and further C-terminal extended precursors were increased in hypergastrinemic atrophic gastritis. Even though the glycine-extended antral gastrins increased only from 0.7% in controls to 2.2% in the patients, the consequence is striking. Thus, the concentrations of glycine-extended gastrins in serum rose from 5 pmol/L in the controls to 106 pmol/L in patients with atrophic gastritis (Table 1). As the ratio between glycine-extended and amidated gastrin in antral veins and antral tissue is the same (36), the high proportion of glycine-extended gastrins in peripheral serum indicates that glycine-extended component I and gastrin 34 (Figure 4C) are cleared at a slower rate than the corresponding amidated forms. Similar mechanisms may explain the high concentrations of further C-terminal extended gastrins in peripheral serum (Figure 4D), whereas constitutive secretion (37) from the G cells has not been reported so far. In addition to the modifications examined in this study, preprogastrin undergoes two further modifications. First, the N-terminal signal peptide is removed cotranslationally in the rough endoplasmatic reticulum (Figure 1). Our approach does not allow estimates of this signalase activity. Second, serine,, phosphorylation has recently been described (38). Such modification might influence the processing of the amidation site, and consequently play a role in the processing observed in this study. The combination of decreased processing and slow clearance changes the plasma profile markedly. Thus, progastrin normally matures almost completely to bioactive, amidated gastrins, which constitute the predominant products of progastrin in normal plasma (39,40). The changed profile in hypergastrinemic atrophic gastritis therefore has two implications. First, accurate measurements of antral G-cell activity cannot be based on assays that measure only amidated gastrins in plasma. Second, as antral hypersecretion is accompanied for example, by enterochromaffinlike-cell proliferation (18,20-22) and disturbed islet cell growth (17) and secretion (19), it becomes important to examine whether non-
PROGASTRIN
PROCESSING
IN HYPERGASTRINEMIA
1069
amidated progastrin products in plasma may influence these effects or whether they are due solely to cY-carboxyamidated gastrins. Two recent studies have also examined human antral progastrin (41,42).Unfortunately, they are difficult to compare with the present study, because both material and methodology differ considerably. Nevertheless, Del Valle et al. (42)found antral glytine-extended gastrins in concentrations similar to those in our controls (Table 3). The two studies also examined hypersecretory tissue, namely gastrinoma extracts (41,42).The mechanisms behind the increased gastrin synthesis in pancreatic tumor cells and in achlorhydric antral G cells make the results difficult to compare. The only common feature seems to be that the concentration of progastrin generally is increased in tissue with enhanced synthesis. References 1. Gregory RA, Tracy HJ. The constitution and properties of two gastrins extracted from hog antral mucosa. Gut 1964;5:10317. of two “big gastrins” from 2. Gregory RA. Tracy HJ. Isolation Zollinger-Ellison tumour tissue. Lancet 1972;ii:797-9. of two mini-gastrins from 3. Gregory RA. Tracy HJ. Isolation Zollinger-Ellison tumour tissue. Gut 1974;15:683-6. 4. Rehfeld JF. Three components of gastrin in human serum. Biochim Biophys Acta 1972;285:364-72. 5. Yoo OJ, Powell CT, Agarwal K. Molecular cloning and nucleotide sequence of full-length cDNA coding for porcine gastrin. Proc Nat1 Acad Sci USA 1982;79:1049-53. cloning of human 6. Boel E, Vuust J. Norris K. et al. Molecular gastrin cDNA: evidence for evolution of gastrin by gene duplication, Proc Nat1 Acad Sci USA 1983:80:2866-9. 7. Fuller PJ. Stone D, Brand SJ. Molecular cloning and sequencing of a rat preprogastrin complementary deoxyribonucleic acid. Mol Endocrinol 1987;1:306-11. 8. Brand SJ, Klarlund J, Schwartz TW, Rehfeld JF. Biosynthesis of tyrosine O-sulfated gastrins in rat antral mucosa. J Biol Chem 1984:259:13246-52. 9. Hilsted L. Rehfeld JF. Alpha-carboxpamidation of antral progastrin: relation to other post-translational modifications. J Biol Chem 1987;262:16953-7. 10. Morley JS. Tracy HJ, Gregory RA. Structure-function relationships in the active C-terminal tetrapeptide sequence of gastrin. Nature 1965;207:1356-9. 11 Jensen SL. Rehfeld JF, Holst JJ, Fahrenkrug J. Nielsen OV, Schaffalitzky de Muckadell OB. Secretory effects of the gastrins on the isolated perfused porcine pancreas. Am J Physiol 1980;238:E186-92. 12 Hilsted L, Hint K. Christiansen J. Rehfeld JF. Neither glycineextended gastrin nor the l-13 fragment of gastrin-17 influences gastric acid secretion in humans. Gastroenterology 1988;94:96-102. 13 De Magistris L. Rehfeld JF. A simple enzymatic procedure for radioimmunochemical quantitation of the large molecular forms of gastrin and cholecystokinin Anal Biochem 1980; 102:126-33. 14. Rehfeld JF, de Magistris L, Andersen BN. Sulfation of gastrin: effect on immunoreactivity. Regul Pept 1981:2:333-42.
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Received March 15, 1988. Accepted November 10, 1988. Address requests for reprints to: Jens F. Rehfeld, M.D.. Department of Clinical Chemistry. Rigshospitalet, DK-2100, Copenhagen, Denmark. This study was supported by grants from the Danish Medical Research Council, and the Lundbeck and Willumsen Foundations. The authors thank Aase Valsted and Lisbeth Andreasen for skillful technical assistance.