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Journal of Physioogy (1991), 433, pp. 483-493 With 5 figure8 Printed in Great Britain
REVERSAL BY OMEPRAZOLE OF THE DEPRESSION OF GASTRIN CELL FUNCTION BY FASTING IN THE RAT
BY R. DIMALINE, D. EVANS, A. VARRO AND G. J. DOCKRAY From the MRC Secretory Control Group, Department of Physiology, University of Liverpool, Liverpool L69 3BX (Received 22 May 1990) SUMMARY
1. Gastrin (G)-cell function is controlled by gastric acid, which has inhibitory effects, and food in the gastric lumen, which has stimulatory effects. We have examined the role of acid in mediating the depression of G-cell function that occurs in fasting in the rat. 2. Rats were fasted for 48 h, and received either the H+-K+-ATPase inhibitor omeprazole, to reduce acid secretion, or vehicle. Basal acid secretion was not significantly different after fasting for 24 or 48 h. Fasted rats which received omeprazole were achlorhydric. 3. In rats treated with vehicle and fasted for 48 h, plasma and tissue gastrin concentrations were significantly depressed. The fall in both parameters suggests an inhibition of gastrin synthesis and consistent with this a decrease was observed in tissue gastrin mRNA abundance and in phosphorylation of progastrin-derived
peptides. 4. In fasted rats treated with omeprazole, tissue gastrin concentrations were not significantly different from those of rats fed ad libitum, but plasma gastrin concentrations were significantly higher than in rats fed ad libitum. Gastrin mRNA abundance and the phosphorylation of progastrin-derived peptides in omeprazoletreated rats was not significantly different from rats fed ad libitum. 5. The data suggest that the depression of G-cell function which occurs in fasted rats can be attributed to the inhibitory action of intraluminal acid on the G-cell. Gastric acid appears to regulate several different aspects of G-cell function, including gastrin synthesis, post-translational processing and secretion. INTRODUCTION
The release of the acid-stimulating hormone gastrin is controlled by the luminal contents of the stomach; food, particularly protein, in the gastric lumen stimulates gastrin release and acid inhibits it (see Dockray & Gregory, 1989). The withdrawal of food from rats for periods of more than 24 h not surprisingly produces a fall in plasma gastrin, but in addition there is also a fall in tissue gastrin concentrations which suggests that gastrin synthesis is depressed (Lichtenberger, Lechago & Johnson, 1975; Track, Creutzfeldt, Arnold & Creutzfeldt, 1978; Mortensen, Morris & MS 8516 16-2
R. DIMALINE, D. EVANS, A. VARRO AND G. J. DOCKRAY Owens, 1979). It is not clear whether the withdrawal of food is by itself sufficient to account for the inhibition of gastrin (G)-cell function induced by fasting, or whether other inhibitory factors are involved. In this context it seems possible that the continued presence of acid in the empty stomach might exert an unrestrained inhibition of the G-cell. In the present study we have tested this idea by using the H+-K+-ATPase inhibitor omeprazole to suppress acid secretion in fasted rats. We have examined the concentrations of gastrin in plasma and in tissue extracts. In order to gain an insight into possible changes in gastrin biosynthesis we also measured gastrin mRNA using a probe generated by the polymerase chain reaction; biologically active gastrins are produced from inactive precursors, and in order to test the idea that withdrawal of food might inhibit this conversion we used in radioimmunoassay two antibodies, one reacting with C-terminally-amidated biologically active gastrins and the other with a peptide corresponding to the extreme C-terminus of the gastrin precursor. Immunoreactivity with the latter type of antibody does not depend on post-translational processing of the precursor (Varro, Desmond, Pauwels, Gregory, Young & Dockray, 1988; Varro, Nemeth, Bridson, Lonovics & Dockray, 1990 b). The peptides produced from the extreme C-terminus of rat progastrin occur in forms differing in phosphorylation and sulphation states, and there is evidence to suggest that decreased phosphorylation is associated with a failure to complete the amidation step required for the biological activity of gastrin (Varro et al. 1990b); accordingly therefore we measured the phosphorylation of progastrin-derived peptides. The results presented here suggest that in fasted rats there is depression of gastrin synthesis, processing and release and that these are a consequence of the inhibitory effects of gastric acid. 484
METHODS
Animals. Studies were made on Wistar rats weighing 210-270 g at the start of the experiment. They were maintained on a 12 h light-dark cycle, and were fed a diet of standard laboratory rat chow (rat and mouse diet, Bantin & Kingman, Hull). Rats were housed on a sawdust floor cage, but during periods of fasting they were transferred to individual wire bottomed cages. Treatments. In one group of rats a gastric cannula was installed to allow determination of acid secretion. These rats were anaesthetized with halothane, followed by pentobarbitone (60 mg kg-', I.P.); a Gregory cannula was fitted in the body of the stomach as described by Dimaline, Carter & Barnes (1986). Three groups of rats were used for studies of gastrin in plasma and tissue, and gastrin mRNA. One group of control animals was fed ad libitum on normal rat chow and allowed free access to water. Two groups of animals were fasted for 48 h, but allowed free access to water; one of these groups received vehicle in the form of 3 ml of 0'25 % (w/v) methyl cellulose (by gavage) at the start of the experimental period and 24 h later. The other group of fasted rats received omeprazole (Astra, UK, 400 ,smol kg-', by gavage) in the form of a suspension in 0-25 % methyl cellulose at the start of the fasting period and 24 h later. Animals used for studies of tissue and plasma gastrin were killed by decapitation 48 h after starting the experiment. Trunk blood was then collected into heparinized tubes for assay of circulating gastrin, centrifuged without delay and the plasma stored at -30 'C. The pyloric antrum was excised by transection at the pylorus and the antro-corpus border. The antrum was then divided by cuts along the lesser and greater curvatures; one sample of antrum was extracted for assay of tissue gastrin immunoreactivity and the other for gastrin mRNA. Tissues to be extracted for immunoreactive gastrin were immediately frozen on dry ice; tissues for extraction of mRNA were processed immediately. Acid secretion studies. Basal acid secretion was collected in rats with a gastric cannula as described by Dimaline et al. (1986). Rats were fasted for 48 h (free access to water) and secretory studies made after 24 h and at the end of the fasting period. One week later the rats were fasted
G-CELL CONTROL MECHANISMS
485
again but received omeprazole (see above) at the start of the period of fasting. Secretory studies were carried out 24 h later. Before secretory studies the gastric cannulae were opened and the stomach gently flushed with warm saline to remove debris, which consisted of hair, mucus particles and occasional pieces of sawdust. Gastric secretions were then collected for six consecutive 15 min ~4-
-~~~
cDNA
Progastrin CFP
L304 P S
G34 G17
M S
Fig. 1. Schematic representation of gastrin cDNA showing the primers (horizontal arrows) used for PCR, and the antibodies used for radioimmunoassay. The cDNA sequence in the box represents the portion complementary to the translated sequence. Progastrin corresponds to the C-terminal 22-104 sequence of the initial precursor and following cleavage, and amidation yields a C-terminal flanking peptide (CFP) and amidated gastrins such as G34 and G17, the latter being by far the most important. The antibody (L2) used to assay G17 reacts with all the main amidated products. The progastrin C-terminal flanking peptide occurs as sulphated (5) and phosphorylated variants (P), which are not distinguished by antibody L304.
periods; acid was determined by titration of aliquots to pH 7-0 with 20 mM-NaOH using a Radiometer automatic titrator. Determination of ga8trin immunoreactivity. Plasma gastrin concentrations were measured by radioimmunoassay using a rabbit antiserum (L2) raised to porcine heptadecapeptide gastrin (G17) and specific for the C-terminus of this molecule. The assay used 126I-labelled human G17. The assay characteristics and the production and characterization of the antiserum have been described in detail by Dockray, Best & Taylor (1977). Concentrations of plasma gastrin were read from a standard curve of synthetic rat unsulphated G17 which was prepared in an appropriate volume of rat plasma previously stripped of gastrin by adsorption of charcoal and filtered through a Sep-pak C18 cartridge. The concentration of gastrin in antral tissue was determined by assay of extracts prepared by boiling tissue in water. Deep frozen tissue (see above) was weighed whilst still frozen and added to vigorously boiling water. Tissues were boiled for 5 min, homogenized and the extracts centrifuged at 2000 g for 10 min; the supernatants were stored at -20 °C prior to assay. This method recovers approximately 90% of total tissue stores of intact progastrin, G17 and C-terminal fragments of progastrin (Desmond, Pauwels, Varro, Gregory, Young & Dockray, 1987; Dockray, Varro, Desmond, Young, Gregory & Gregory 1987). In radioimmunoassay, tissue extracts were diluted to give inhibition of binding of label to antiserum in the mid-region of the standard curve. Two different assay systems were used; one employed antiserum L2 as described above. The other assay used a rabbit antiserum (L304) raised to the C-terminal tryptic peptide of rat progastrin (Fig. 1). This assay reads intact rat progastrin and C-terminal fragments of it generated by tryptic-like cleavage during post-translational processing to yield biologically active G17. The C-terminal fragment of rat progastrin exists in several forms differing in whether or not Ser94 is phosphorylated and Tyr103 sulphated (Varro, Bridson, Nemeth & Dockray, 1990a). Antibody L304 reacts with all these variants; in the present study an estimate of the relative concentrations of different variants was obtained by fractionation of tissue extracts by ion exchange chromatography on DEAE cellulose as previously described (Varro et al. 1990b). This system resolves, in order of elution, the unmodified C-terminal tryptic peptide of progastrin, the phosphorylated (unsulphated) peptide, the sulphated (unphosphorylated) peptide and both phosphorylated and sulphated peptide. Gastrin mRNA. The oligonucleotides used to prepare probes for gastrin mRNA determination
R. DIMALINE, D. EVANS, A. VARRO AND G. J. DOCKRAY were synthesized using an Applied Biosystems 391 DNA synthesizer (Applied Biosystems, Warrington, Cheshire) and purified on an oligonucleotide purification cartridge. They corresponded to bases -18 to +4 (sense strand) and bases 344-325 (antisense strand) of the rat gastrin cDNA
486
sequence (Fuller, Stone & Brand, 1987) (Fig. 1). Total RNA from rat antrum was extracted by a
Origin -
434
_
267-
Fig. 2. The rat gastrin cDNA probe generated by the polymerase chain reaction (PCR) as described in the methods. An aliquot of the PCR reaction was electrophoresed in a 1-5 % agarose gel containing ethidium bromide. The gel was exposed1 to Kodak X-AR film for 40 min. Arrows indicate the origin and the position of restriction fragment markers from a total digest of pBR322 DNA by Hae III (Sigma). There is a single band of 32P-labelled gastrin PCR product with a molecular weight compatible with the predicted value (362 base pairs).
modification of the method of Chirgwin, Przbyla, MacDonald & Rutter (1979). Samples of antral cDNA were prepared using a Boehringer Mannheim cDNA synthesis kit and used as templates for the polymerase chain reaction (PCR) employing a Perkin Elmer-Cetus Gene-Amp kit. Incorporation of 32P into the amplified product was achieved .by substituting 98% of the deoxycytidine triphosphate (dCTP) in the PCR reaction with 100 ,uCi [32P] dCTP (Amersham). The radiolabelled cDNA probes were purified on NAP 5 columns (Pharmacia) and denatured before hybridization by boiling for 5 min. Probes were analysed by electrophoresis in 1P5 % agarose gels run in Tris-borate-EDTA buffer, which revealed a product of 362 base pairs that corresponded to the predicted size of the PCR product (Fig. 2). For assay of gastrin mRNA, samples of antral RNA were extracted as described above, quantified by absorbance at 260 nm and applied to 1 % agarose formaldehyde gels containing ethidium bromide. Gels were electrophoresed in MOPS running buffer (0-02 M) at 60 V for about 3 h, and RNA electrotransferred onto nylon membranes (Hybond N, Amersham) and cross-linked with ultraviolet light. Membranes were incubated for 6 h at 42 °C in prehybridization buffer (50 % formamide, 5 x Denhardt's solution, 5 x SSPE (standard sodium phosphate EDTA), 0 5% SDS (sodium dodecyl sulphate) and 200 jug ml-' sonicated salmon sperm DNA) then for 18 h in the same solution containing gastrin cDNA probe (2 x 106 c.p.m. ml-'). Membranes were washed twice for 20 min at room temperature in 2 x SSPE containing 0-1 % SDS, and once for 20 min at 65 °C in 0-1 x SSPE containing 0-1 % SDS. Hybridized membranes were exposed to Kodak X-AR film for 48 h at -80 °C using an intensifying screen. The bands corresponding to gastrin mRNA were quantified using a scanning laser densitometer. Stati8tic8. Results are expressed as means+ s.E.M. and comparisons -were made by t test. RESULTS
Acid secretion The basal acid secretion in rats fasted for 24 h was comparable to that described in previous studies, i.e. 120-150 #tmol h-' (Dimaline et al. 1986). After 48 h fasting the basal secretion was not significantly different from that at 24 h (Table 1).
G-CELL CONTROL MECHANISMS
487
400
e300 C
0)
200-
40)
C
100 0j Co~~~~~~~~~
E
z
0-
E
.2 0
0~
Fig. 3. Relative changes in plasma gastrin and tissue gastrin measured with antibody L2, tissue gastrin mRNA and progastrin-derived peptide phosphorylation in fasted and omeprazole-treated rats. The data are expressed as a percentage of the values for rats fed ad libitum shown as open bars; for each parameter values for fasted vehicle-treated rats are shown to the left of those for fed rats, and values for fasted omeprazole-treated rats are shown to the right. Note that fasting for 48 h significantly reduced each parameter (P < 0-05). In fasted rats that were treated with omeprazole, plasma gastrin was significantly higher than in fasted or fed rats, but phosphorylation, gastrin mRNA and tissue gastrin concentrations were not significantly different. All values are means and vertical bars are S.E.M., for each group.
TABLE 1. Basal acid secretion 24 h fasted Sample
(,smol 15 min)-' in fasted rats* 48 hr fasted
24 h + omeprazole
1 32A4+2-3 t 33A4±6-5 2 t 31P5±7-2 25-3±5A4 3 34-8+7-8 32-1+4-9 t 4 29-5±5 9 35-5±3-2 t 5 32-9+4-4 30-2+3-8 t 6 25-7+5-9 34-2+4-6 t * Acid secretion was measured in six consecutive 15 min periods in the same six rats fasted for 24 h and again at 48 h. On a separate occasion acid secretion was measured after treatment of the rats with omeprazole at the start of the 24 h fast. t In five of six rats all samples had a pH > 7 0; one rat secreted 012-0O42 lsmol (15 min)-' for three of six periods, and for the other three periods was achlorhydric.
Treatment of the same rats with omeprazole strongly inhibited acid secretion. Thus 24 h after receiving omeprazole, five of six rats were achiorhydric, and one rat produced < 2-5 ,umol h-'.
Plasma gastrin The concentration of plasma gastrin in control rats fed ad libitum was 500 + 4-8 pmol 1-1; fasting for 48 h reduced concentrations to 14-3 + 4-3 pmol 1-1 (P < 005) (Fig. 3). In rats fasted for 48 h and treated with omeprazole the circulating
R. DIMALINE, D. EVANS, A. VARRO AND G. J. DOCKRAY gastrin concentration was 142 + 36 pmol 1', which was significantly greater than that in either fed ad libitum or fasted vehicle-treated rats (P < 001).
488
Tissue gastrin The concentrations of immunoreactive gastrin in the antral tissue of rats fed ad libitum measured with antibody L2 were 1P25 + 017 nmol g-i, and these were reduced
I -
E E
'a
:t
20 40 60 80 100 120140 Elution volume (ml)
Fig. 4. Separation by DEAE ion exchange chromatography of different forms of progastrin-derived peptides measured with antibody L304. In both fasted (A) and fastedomeprazole treated rats (B) four peaks were consistently separated. These correspond to progressively more acidic peptides and in order of elution are (a) the unmodified peptide (CFP-I), (b) phosphorylated (CFP-II), (c) sulphated (CFP-III) and (d) both phosphorylated and sulphated peptides (CFP-IV). See text for estimates of the proportion of progastrin-derived peptides in the phosphorylated state.
by about 50% (0-67 + 011 nmol g-1) in rats fasted for 48 h. In fasted rats treated with omeprazole, however, tissue concentrations of gastrin measured with L2 were not significantly different from those in rats fed ad libitum (099 + 020 nmol g'). Assays using antibody L304 which is specific for the C-terminus of progastrin recorded rather higher concentrations of immunoreactivity compared with antibody L2, in all three groups of rats (Table 2). The same pattern was observed, however, in that in fasted rats there were significantly decreased tissue conoentrations compared with rats fed ad libitum, while in rats treated with omeprazole concentrations were not significantly reduced.
G-CELL CONTROL MECHANISMS Lane
489
7 8 9 10 11
1 2 3 4 5 6
A 28 s18 s--
Lane
1 2 3 4 5 6
7 8 9 10 1112
B 28 s_ 18 s -_-
Fig. 5. A, northern blot analysis of gastrin mRNA levels in 20 ,ug samples of total antral RNA from rats fasted for 48 h (lanes 1-6) or fed ad libitum (lanes 7-11). Arrows indicate the positions of ribosomal subunits. B, Northern blot analysis of gastrin mRNA levels in 20 jug samples of total antral mRNA from rats fasted for 48 h (lanes 1-6) or fasted for 48 h with omeprazole treatment (7-12). TABLE 2. Tissue and plasma gastrin concentrations in fed and fasted rats (mean ± s.E.M., n=
Plasma gastrin
Group Fed ad libitum Fasted, vehicle Fasted, omeprazole
(pmol l-1) 50 0+4-8
6)*
Tissue gastrin (L2) Tissue gastrin (L304) (nmol g-1) (nmol g-1)
1P25+0417
1P75+0 25
14-4 + 4-3t 0.67 + 011 t 1P06 ± 041I t 142 + 36t 0-99+0-20 1P31+ 0419 * Plasma gastrin was measured with antibody L2 specific for the C-terminal of G17. Tissue gastrin concentration was measured with this antibody and with antibody L304 which is specific for the C-terminal fragment of rat progastrin. t Significantly different from rats fed ad libitum (P < 0 05)
The C-terminal fragments of progastrin that react with antibody L304 occur in several different forms that can be separated by ion exchange chromatography. In order of elution from DEAE these are unmodified, phosphorylated, sulphated, and both phosphorylated and sulphated (Fig. 4). Fasting decreased the proportion of progastrin-derived peptides that occurs in the phosphorylated state; thus in rats fed ad libitum, 51-6 + 4-4% of progastrin-derived C-terminal flanking peptide was phosphorylated compared with 33-0+444% in rats which were fasted and treated with vehicle (P < 0 05). In fasted rats treated with omeprazole the phosphorylation of progastrin-derived peptides (52-2 + 2-9 %) was not significantly different from that in rats fed ad libitum. The proportion of progastrin-derived C-terminal flanking peptide that was sulphated (approximately 90%) was not significantly changed by fasting.
490
R. DIMALINE, D. EVANS, A. VARRO AND G. J. DOCKRAY
mRNA Northern blots of gastrin mRNA revealed a single band in both fasted and fed rats that was compatible with an mRNA species of 650 bases. The quantification of mRNA by scanning laser densitometry indicated that the abundance of gastrin mRNA in fasted rats was 65-6 + 107 % of that in rats fed ad libitum (P < 005) (Fig. 5). In contrast, in fasted rats treated with omeprazole gastrin mRNA was 134'3+22-1 % of that in rats fed ad libitum (Fig. 5). DISCUSSION
The decrease in tissue and plasma gastrin concentrations that occurs on withdrawal of food in the rat has been described by several groups (Lichtenberger et al. 1975; Track et al. 1978; Mortensen et al. 1979), and is confirmed in the present study. In addition, in fasting there is (a) a depression of tissue gastrin mRNA levels which suggests either depressed gastrin gene transcription or increased mRNA degradation, and (b) a decrease in the proportion of progastrin-derived peptides that occur in the phosphorylated state, which is a marker for progastrin processing. Together the data indicate that in fasted rats, both gastrin release and biosynthesis are reduced. The primary observation of the present study is that omeprazole reverses these changes in G-cell function. The major luminal factors that control pyloric antral G-cells are protein in the gastric lumen (which stimulates) and acid (which inhibits). The H+-K+-ATPase inhibitor omeprazole is a powerful inhibitor of acid secretion. The dose we used was shown to produce achlorhydria for at least 24 h. It is known that in rats fed ad libitum similar doses of omeprazole produce hypergastrinaemia, increased gastrin mRNA and eventually G-cell hyperplasia (Allen, Bishop, Daly, Larsson, Carlsson, Polak & Bloom 1986; Larsson, Carlsson, Mattsson, Lundell, Sundler, Sundell, Wallmark, Watanabe & Hakanson, 1986; Brand & Stone, 1988). The demonstration that omeprazole reverses the inhibitory effects of fasting on the G-cell suggests that the latter are attributable to the unrestrained inhibition of G-cell function by luminal acid. It may be that the rat is particularly susceptible to the inhibitory effects of acid in the fasting state because basal rates of secretion are relatively high. Thus when normalized to body weight, basal acid secretion in the rat is 400-600 #smol kg-' h-'; and is 20-30 % of maximal acid output, whereas in dog and man, basal acid output is lower whether expressed relative to body weight (5-40 #nmol kg-' h-') or as a proportion of maximal, i.e. 1-10 % (Debas, 1987). Rates of acid secretion after 48 h fasting in rat were similar to those at 24 h, and to those found after an overnight fast (approximately 15 h). Since plasma gastrin decreased sharply with 48 h fasting it would appear that it is not responsible for maintaining basal acid secretion. There is now good evidence that acid releases somatostatin from pyloric antral D-cells which in turn acts as a paracrine inhibitor of gastrin release and gastrin synthesis (Saffouri, Weir, Bitar & Makhlouf, 1980; Short, Doyle & Wolfe, 1984; Brand & Stone, 1988; Dockray & Gregory, 1989; Karnik, Monahan & Wolfe, 1989). It seems possible, then, that in the absence of stimulation by food basal acid secretion in fasted rats releases somatostatin which acts to depress gastrin synthesis
G-CELL CONTROL MECHANISMS 491 and release. These data imply that there is tonic inhibition of the rat G-cell by acid. Previous studies in other species (dog, man) have generally failed to demonstrate that acute alkalinization of the empty stomach increases plasma gastrin (Walsh & Grossman, 1975). Whether or not maintained alkalinization of the empty stomach for 48 h, as in this study, influences human or dog G-cell function remains to be seen. For reasons mentioned above, the rat may be the species of choice for demonstrating the tonic inhibitory effects of gastric acid on G-cells. Protein or peptide synthesis is in principle controlled at several levels including mRNA transcription, mRNA degradation, translational rates and post-translational processing. The data on gastrin mRNA levels provide clear evidence that either transcription is depressed or degradation increased with fasting; in the dog there is evidence that somatostatin affects both (Karnik et al. 1989). Antibody L2 reacts with biologically active C-terminally amidated gastrin, and would not reveal inactive precursors of intermediates; on its own therefore assays with this antibody cannot provide much insight into the control of post-translational processing. However, antibody L304 reacts both with progastrin and its C-terminal fragment which means it can detect the products of gastrin mRNA translation, regardless of subsequent processing. In many species there is a 1:1 stoichiometry between amidated gastrin and progastrin C-terminal fragments (Dockray & Gregory, 1989). In the rat, however, there are appreciable quantities of a Gly-extended .biosynthetic intermediate, which is the immediate precursor of the amidated gastrins (Daugherty & Yamada, 1989). The lower tissue concentrations measured with L2 compared with L304 can be accounted for by the failure to convert fully Gly-extended intermediates to G17. Assays with antibody L304 showed a depression in tissue concentrations of immunoreactivity comparable to those with L2, which indicates depressed production of progastrin, and so allows us to exclude the possibility that the low tissue and plasma gastrin in fasted rats is attributable to a failure to convert inactive precursor forms to biologically active amidated gastrins. The C-terminal fragment of progastrin is phosphorylated in many species (Dockray et al. 1987; Desmond, Varro, Young, Gregory, Nemeth & Dockray, 1989), and there is evidence that depressed phosphorylation is associated with decreased synthesis (Varro et al. 1990b). The finding of reduced phosphorylation of progastrin-derived peptides in fasted rats contributes to the idea that phosphorylation provides an index of gastrin biosynthetic activity. More generally, however, the reversal of phosphorylation in omeprazole-treated rats is direct evidence that at least some of the post-translational processing events in gastrin biosynthesis are regulated by the gastric luminal contents. It is well recognized that prolonged achlorhydria in ad libitum fed animals leads to G-cell hyperplasia, hypergastrinaemia, and increased tissue gastrin mRNA (see Dockray & Gregory, 1989). Our data indicate that in the rat, inhibition of acid alone is sufficient to produce the same effect, i.e. direct stimulation by food is not necessary for maintenance of plasma gastrin or of gastrin synthesis rates. In the present experiment we did not estimate G-cell numbers, but since these cells turn over slowly we assume the G-cell population did not change (Thompson, Price & Wright, 1990). The data strongly suggest that luminal acid is able to work acutely at three levels to control G-cell function, namely by regulating progastrin mRNA abundance,
492
R. DIMALINE, D. EVANS, A. VARRO AND G. J. DOCKRAY
progastrin post-translational processing and gastrin secretion. Since plasma gastrin concentrations in omeprazole-treated rats exceeded those in fed ad libitum rats it would appear that depression by acid is actually more important than stimulation by food for controlling the secretory activity of rat G-cells. We are grateful to the MRC for financial support, to Christine Carter for help in preparing the manuscript, and to Dr D. Edgar for the use of a scanning laser densitometer. REFERENCES
ALLEN, J. M., BISHOP, A. E., DALY, M. J., LARSSON, H., CARLSSON, E., POLAK, J. M. & BLOOM, S. R. (1986). Effect of inhibition of acid secretion on the regulatory peptides in the rat stomach. Gastroenterology 90, 970-977. BRAND, S. J. & STONE, D. (1988). Reciprocal regulation of antral gastrin and somatostatin gene expression in omeprazole-induced achlorhydria. Journal of Clinical Investigation 82, 1059-1066. CHIRGWIN, J. J., PRZBYLA, A. E., MACDONALD, R. J. & RUTTER, W. J. (1979). Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18, 5294-5299.
DAUGHERTY, D. & YAMADA, T. (1989). Posttranslational processing of gastrin. Physiological Reviews 69, 482-502. DEBAS, H. T. (1987). Peripheral regulation of gastric acid secretion. In Physiology of the Gastrointestinal Tract, 2nd edn, ed. JOHNSON, L. R., pp. 931-945. Raven Press, New York. DESMOND, H., PAUWELS, S., VARRO, A., GREGORY, H., YOUNG, J. & DOCKRAY, G. J. (1987). Isolation and characterization of the intact gastrin precursor from a gastrinoma. FEBS Letters
210, 185-188. DESMOND, H., VARRO, A., YOUNG, J., GREGORY, H., NEMETH, J. & DOCKRAY, G. J. (1989). The constitution and properties of phosphorylated and unphosphorylated C-terminal fragments of progastrin from dog and ferret antrum. Regulatory Peptides 25, 223-233. DIMALINE, R., CARTER, N. & BARNES, S. (1986). Evidence for reflex adrenergic inhibition of acid secretion in the conscious rat. American Journal of Physiology 251, G615-618. DOCKRAY, G. J., BEST, L. & TAYLOR, I. L. (1977). Immunochemical characterization of gastrin in pancreatic islets of normal and genetically obese mice. Journal of Endocrinology 72, 143-151. DOCKRAY, G. J. & GREGORY, R. A. (1989). Gastrin. In Handbook of Physiology - The Gastrointestinal System II, chap. 15, ed. MAKHLOUF, G. M., pp. 311-336. American Physiological Society, Bethesda, MD, USA. DOCKRAY, G. J., VARRO, A., DESMOND, H., YOUNG, J., GREGORY, H. & GREGORY, R. A. (1987). Post-translational processing of the porcine gastrin precursor by phosphorylation of the COOHterminal fragment. Journal of Biological Chemistry 262, 8643-8647. FULLER, P. J., STONE, D. L. & BRAND, S. J. (1987). Molecular cloning and sequencing of a rat preprogastrin complementary deoxyribonucleic acid. Molecular Endocrinology 1, 306-311. KARNIK, P. S., MONAHAN, S. J. & WOLFE, M. (1989). Inhibition of gastrin gene expression by somatostatin. Journal of Clinical Investigation 83, 367-372. LARSSON, H., CARLSSON, E., MATTSSON, H., LUNDELL, L., SUNDLER, F., SUNDELL, G., WALLMARK, B., WATANABE, T. & HAKANSON, R. (1986). Plasma gastrin and gastric enterochromaffinlike cell activation and proliferation: Studies with omeprazole and ranitidine in intact and antrectomized rats. Gastroenterology 90, 391-399. LICHTENBERGER, L. M., LECHAGO, J. & JOHNSON, L. R. (1975). Depression of antral and serum gastrin concentration by food deprivation in the rat. Gastroenterology 68, 1473-1479. MORTENSEN, N. J. MCC., MORRIS, J. F. & OWENS, C. (1979). Gastrin and the ultrastructure of G cells in the fasting rat. Gut 20, 41-50. SAFFOURI, B., WEIR, G. C., BITAR, K. N. & MAKHLOUF, G. M. (1980). Gastrin and somatostatin secretion by perfused rat stomach: Functional linkage of antral peptides. American Journal of
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THOMPSON, E. M., PRICE, Y. E. & WRIGHT, N. A. (1990). Kinetics of enteroendocrine cells with implications for their origin: a study of the cholecystokinin and gastrin subpopulations combining tritiated thymidine labelling with immunocytochemistry in the mouse. Gut 31, 406 411. TRACK, N. S., CREUTZFELDT, C., ARNOLD, R. & CREUTZFELDT, W. (1978). The antral gastrinproducing G-cell: Biochemical and ultrastructural responses to feeding. Cell and Tissue Research 194, 131-139. . VARRO, A., BRIDSON, J., NEMETH, J. & DOCKRAY, G. J. (1990a). The influence of feeding and fasting on the phosphorylation of gastrin-related peptides in the pyloric antral mucosa of the rat. Journal of Physiology 424, 1 IP. VARRO, A., DESMOND, H., PAUWELS, S., GREGORY, H., YOUNG, J. & DOCKRAY, G. J. (1988). The human gastrin precursor: Characterization of phosphorylated forms and fragments. Biochemical Journal 256, 951-957. VARRO, A., NEMETH, J., BRIDSON, J., LoNovIcs, J. & DOCKRAY, G. J. (1900 b). Modulation of posttranslational processing of gastrin precursor in dogs. American Journal of Physiology (in the Press). WALSH, J. H. & GROSSMAN, M. I. (1975). Gastrin. New England Journal ofMedicine 292, 1324-1332.
Journal of Physioogy (1991), 433, pp. 483-493 With 5 figure8 Printed in Great Britain
REVERSAL BY OMEPRAZOLE OF THE DEPRESSION OF GASTRIN CELL FUNCTION BY FASTING IN THE RAT
BY R. DIMALINE, D. EVANS, A. VARRO AND G. J. DOCKRAY From the MRC Secretory Control Group, Department of Physiology, University of Liverpool, Liverpool L69 3BX (Received 22 May 1990) SUMMARY
1. Gastrin (G)-cell function is controlled by gastric acid, which has inhibitory effects, and food in the gastric lumen, which has stimulatory effects. We have examined the role of acid in mediating the depression of G-cell function that occurs in fasting in the rat. 2. Rats were fasted for 48 h, and received either the H+-K+-ATPase inhibitor omeprazole, to reduce acid secretion, or vehicle. Basal acid secretion was not significantly different after fasting for 24 or 48 h. Fasted rats which received omeprazole were achlorhydric. 3. In rats treated with vehicle and fasted for 48 h, plasma and tissue gastrin concentrations were significantly depressed. The fall in both parameters suggests an inhibition of gastrin synthesis and consistent with this a decrease was observed in tissue gastrin mRNA abundance and in phosphorylation of progastrin-derived
peptides. 4. In fasted rats treated with omeprazole, tissue gastrin concentrations were not significantly different from those of rats fed ad libitum, but plasma gastrin concentrations were significantly higher than in rats fed ad libitum. Gastrin mRNA abundance and the phosphorylation of progastrin-derived peptides in omeprazoletreated rats was not significantly different from rats fed ad libitum. 5. The data suggest that the depression of G-cell function which occurs in fasted rats can be attributed to the inhibitory action of intraluminal acid on the G-cell. Gastric acid appears to regulate several different aspects of G-cell function, including gastrin synthesis, post-translational processing and secretion. INTRODUCTION
The release of the acid-stimulating hormone gastrin is controlled by the luminal contents of the stomach; food, particularly protein, in the gastric lumen stimulates gastrin release and acid inhibits it (see Dockray & Gregory, 1989). The withdrawal of food from rats for periods of more than 24 h not surprisingly produces a fall in plasma gastrin, but in addition there is also a fall in tissue gastrin concentrations which suggests that gastrin synthesis is depressed (Lichtenberger, Lechago & Johnson, 1975; Track, Creutzfeldt, Arnold & Creutzfeldt, 1978; Mortensen, Morris & MS 8516 16-2
R. DIMALINE, D. EVANS, A. VARRO AND G. J. DOCKRAY Owens, 1979). It is not clear whether the withdrawal of food is by itself sufficient to account for the inhibition of gastrin (G)-cell function induced by fasting, or whether other inhibitory factors are involved. In this context it seems possible that the continued presence of acid in the empty stomach might exert an unrestrained inhibition of the G-cell. In the present study we have tested this idea by using the H+-K+-ATPase inhibitor omeprazole to suppress acid secretion in fasted rats. We have examined the concentrations of gastrin in plasma and in tissue extracts. In order to gain an insight into possible changes in gastrin biosynthesis we also measured gastrin mRNA using a probe generated by the polymerase chain reaction; biologically active gastrins are produced from inactive precursors, and in order to test the idea that withdrawal of food might inhibit this conversion we used in radioimmunoassay two antibodies, one reacting with C-terminally-amidated biologically active gastrins and the other with a peptide corresponding to the extreme C-terminus of the gastrin precursor. Immunoreactivity with the latter type of antibody does not depend on post-translational processing of the precursor (Varro, Desmond, Pauwels, Gregory, Young & Dockray, 1988; Varro, Nemeth, Bridson, Lonovics & Dockray, 1990 b). The peptides produced from the extreme C-terminus of rat progastrin occur in forms differing in phosphorylation and sulphation states, and there is evidence to suggest that decreased phosphorylation is associated with a failure to complete the amidation step required for the biological activity of gastrin (Varro et al. 1990b); accordingly therefore we measured the phosphorylation of progastrin-derived peptides. The results presented here suggest that in fasted rats there is depression of gastrin synthesis, processing and release and that these are a consequence of the inhibitory effects of gastric acid. 484
METHODS
Animals. Studies were made on Wistar rats weighing 210-270 g at the start of the experiment. They were maintained on a 12 h light-dark cycle, and were fed a diet of standard laboratory rat chow (rat and mouse diet, Bantin & Kingman, Hull). Rats were housed on a sawdust floor cage, but during periods of fasting they were transferred to individual wire bottomed cages. Treatments. In one group of rats a gastric cannula was installed to allow determination of acid secretion. These rats were anaesthetized with halothane, followed by pentobarbitone (60 mg kg-', I.P.); a Gregory cannula was fitted in the body of the stomach as described by Dimaline, Carter & Barnes (1986). Three groups of rats were used for studies of gastrin in plasma and tissue, and gastrin mRNA. One group of control animals was fed ad libitum on normal rat chow and allowed free access to water. Two groups of animals were fasted for 48 h, but allowed free access to water; one of these groups received vehicle in the form of 3 ml of 0'25 % (w/v) methyl cellulose (by gavage) at the start of the experimental period and 24 h later. The other group of fasted rats received omeprazole (Astra, UK, 400 ,smol kg-', by gavage) in the form of a suspension in 0-25 % methyl cellulose at the start of the fasting period and 24 h later. Animals used for studies of tissue and plasma gastrin were killed by decapitation 48 h after starting the experiment. Trunk blood was then collected into heparinized tubes for assay of circulating gastrin, centrifuged without delay and the plasma stored at -30 'C. The pyloric antrum was excised by transection at the pylorus and the antro-corpus border. The antrum was then divided by cuts along the lesser and greater curvatures; one sample of antrum was extracted for assay of tissue gastrin immunoreactivity and the other for gastrin mRNA. Tissues to be extracted for immunoreactive gastrin were immediately frozen on dry ice; tissues for extraction of mRNA were processed immediately. Acid secretion studies. Basal acid secretion was collected in rats with a gastric cannula as described by Dimaline et al. (1986). Rats were fasted for 48 h (free access to water) and secretory studies made after 24 h and at the end of the fasting period. One week later the rats were fasted
G-CELL CONTROL MECHANISMS
485
again but received omeprazole (see above) at the start of the period of fasting. Secretory studies were carried out 24 h later. Before secretory studies the gastric cannulae were opened and the stomach gently flushed with warm saline to remove debris, which consisted of hair, mucus particles and occasional pieces of sawdust. Gastric secretions were then collected for six consecutive 15 min ~4-
-~~~
cDNA
Progastrin CFP
L304 P S
G34 G17
M S
Fig. 1. Schematic representation of gastrin cDNA showing the primers (horizontal arrows) used for PCR, and the antibodies used for radioimmunoassay. The cDNA sequence in the box represents the portion complementary to the translated sequence. Progastrin corresponds to the C-terminal 22-104 sequence of the initial precursor and following cleavage, and amidation yields a C-terminal flanking peptide (CFP) and amidated gastrins such as G34 and G17, the latter being by far the most important. The antibody (L2) used to assay G17 reacts with all the main amidated products. The progastrin C-terminal flanking peptide occurs as sulphated (5) and phosphorylated variants (P), which are not distinguished by antibody L304.
periods; acid was determined by titration of aliquots to pH 7-0 with 20 mM-NaOH using a Radiometer automatic titrator. Determination of ga8trin immunoreactivity. Plasma gastrin concentrations were measured by radioimmunoassay using a rabbit antiserum (L2) raised to porcine heptadecapeptide gastrin (G17) and specific for the C-terminus of this molecule. The assay used 126I-labelled human G17. The assay characteristics and the production and characterization of the antiserum have been described in detail by Dockray, Best & Taylor (1977). Concentrations of plasma gastrin were read from a standard curve of synthetic rat unsulphated G17 which was prepared in an appropriate volume of rat plasma previously stripped of gastrin by adsorption of charcoal and filtered through a Sep-pak C18 cartridge. The concentration of gastrin in antral tissue was determined by assay of extracts prepared by boiling tissue in water. Deep frozen tissue (see above) was weighed whilst still frozen and added to vigorously boiling water. Tissues were boiled for 5 min, homogenized and the extracts centrifuged at 2000 g for 10 min; the supernatants were stored at -20 °C prior to assay. This method recovers approximately 90% of total tissue stores of intact progastrin, G17 and C-terminal fragments of progastrin (Desmond, Pauwels, Varro, Gregory, Young & Dockray, 1987; Dockray, Varro, Desmond, Young, Gregory & Gregory 1987). In radioimmunoassay, tissue extracts were diluted to give inhibition of binding of label to antiserum in the mid-region of the standard curve. Two different assay systems were used; one employed antiserum L2 as described above. The other assay used a rabbit antiserum (L304) raised to the C-terminal tryptic peptide of rat progastrin (Fig. 1). This assay reads intact rat progastrin and C-terminal fragments of it generated by tryptic-like cleavage during post-translational processing to yield biologically active G17. The C-terminal fragment of rat progastrin exists in several forms differing in whether or not Ser94 is phosphorylated and Tyr103 sulphated (Varro, Bridson, Nemeth & Dockray, 1990a). Antibody L304 reacts with all these variants; in the present study an estimate of the relative concentrations of different variants was obtained by fractionation of tissue extracts by ion exchange chromatography on DEAE cellulose as previously described (Varro et al. 1990b). This system resolves, in order of elution, the unmodified C-terminal tryptic peptide of progastrin, the phosphorylated (unsulphated) peptide, the sulphated (unphosphorylated) peptide and both phosphorylated and sulphated peptide. Gastrin mRNA. The oligonucleotides used to prepare probes for gastrin mRNA determination
R. DIMALINE, D. EVANS, A. VARRO AND G. J. DOCKRAY were synthesized using an Applied Biosystems 391 DNA synthesizer (Applied Biosystems, Warrington, Cheshire) and purified on an oligonucleotide purification cartridge. They corresponded to bases -18 to +4 (sense strand) and bases 344-325 (antisense strand) of the rat gastrin cDNA
486
sequence (Fuller, Stone & Brand, 1987) (Fig. 1). Total RNA from rat antrum was extracted by a
Origin -
434
_
267-
Fig. 2. The rat gastrin cDNA probe generated by the polymerase chain reaction (PCR) as described in the methods. An aliquot of the PCR reaction was electrophoresed in a 1-5 % agarose gel containing ethidium bromide. The gel was exposed1 to Kodak X-AR film for 40 min. Arrows indicate the origin and the position of restriction fragment markers from a total digest of pBR322 DNA by Hae III (Sigma). There is a single band of 32P-labelled gastrin PCR product with a molecular weight compatible with the predicted value (362 base pairs).
modification of the method of Chirgwin, Przbyla, MacDonald & Rutter (1979). Samples of antral cDNA were prepared using a Boehringer Mannheim cDNA synthesis kit and used as templates for the polymerase chain reaction (PCR) employing a Perkin Elmer-Cetus Gene-Amp kit. Incorporation of 32P into the amplified product was achieved .by substituting 98% of the deoxycytidine triphosphate (dCTP) in the PCR reaction with 100 ,uCi [32P] dCTP (Amersham). The radiolabelled cDNA probes were purified on NAP 5 columns (Pharmacia) and denatured before hybridization by boiling for 5 min. Probes were analysed by electrophoresis in 1P5 % agarose gels run in Tris-borate-EDTA buffer, which revealed a product of 362 base pairs that corresponded to the predicted size of the PCR product (Fig. 2). For assay of gastrin mRNA, samples of antral RNA were extracted as described above, quantified by absorbance at 260 nm and applied to 1 % agarose formaldehyde gels containing ethidium bromide. Gels were electrophoresed in MOPS running buffer (0-02 M) at 60 V for about 3 h, and RNA electrotransferred onto nylon membranes (Hybond N, Amersham) and cross-linked with ultraviolet light. Membranes were incubated for 6 h at 42 °C in prehybridization buffer (50 % formamide, 5 x Denhardt's solution, 5 x SSPE (standard sodium phosphate EDTA), 0 5% SDS (sodium dodecyl sulphate) and 200 jug ml-' sonicated salmon sperm DNA) then for 18 h in the same solution containing gastrin cDNA probe (2 x 106 c.p.m. ml-'). Membranes were washed twice for 20 min at room temperature in 2 x SSPE containing 0-1 % SDS, and once for 20 min at 65 °C in 0-1 x SSPE containing 0-1 % SDS. Hybridized membranes were exposed to Kodak X-AR film for 48 h at -80 °C using an intensifying screen. The bands corresponding to gastrin mRNA were quantified using a scanning laser densitometer. Stati8tic8. Results are expressed as means+ s.E.M. and comparisons -were made by t test. RESULTS
Acid secretion The basal acid secretion in rats fasted for 24 h was comparable to that described in previous studies, i.e. 120-150 #tmol h-' (Dimaline et al. 1986). After 48 h fasting the basal secretion was not significantly different from that at 24 h (Table 1).
G-CELL CONTROL MECHANISMS
487
400
e300 C
0)
200-
40)
C
100 0j Co~~~~~~~~~
E
z
0-
E
.2 0
0~
Fig. 3. Relative changes in plasma gastrin and tissue gastrin measured with antibody L2, tissue gastrin mRNA and progastrin-derived peptide phosphorylation in fasted and omeprazole-treated rats. The data are expressed as a percentage of the values for rats fed ad libitum shown as open bars; for each parameter values for fasted vehicle-treated rats are shown to the left of those for fed rats, and values for fasted omeprazole-treated rats are shown to the right. Note that fasting for 48 h significantly reduced each parameter (P < 0-05). In fasted rats that were treated with omeprazole, plasma gastrin was significantly higher than in fasted or fed rats, but phosphorylation, gastrin mRNA and tissue gastrin concentrations were not significantly different. All values are means and vertical bars are S.E.M., for each group.
TABLE 1. Basal acid secretion 24 h fasted Sample
(,smol 15 min)-' in fasted rats* 48 hr fasted
24 h + omeprazole
1 32A4+2-3 t 33A4±6-5 2 t 31P5±7-2 25-3±5A4 3 34-8+7-8 32-1+4-9 t 4 29-5±5 9 35-5±3-2 t 5 32-9+4-4 30-2+3-8 t 6 25-7+5-9 34-2+4-6 t * Acid secretion was measured in six consecutive 15 min periods in the same six rats fasted for 24 h and again at 48 h. On a separate occasion acid secretion was measured after treatment of the rats with omeprazole at the start of the 24 h fast. t In five of six rats all samples had a pH > 7 0; one rat secreted 012-0O42 lsmol (15 min)-' for three of six periods, and for the other three periods was achlorhydric.
Treatment of the same rats with omeprazole strongly inhibited acid secretion. Thus 24 h after receiving omeprazole, five of six rats were achiorhydric, and one rat produced < 2-5 ,umol h-'.
Plasma gastrin The concentration of plasma gastrin in control rats fed ad libitum was 500 + 4-8 pmol 1-1; fasting for 48 h reduced concentrations to 14-3 + 4-3 pmol 1-1 (P < 005) (Fig. 3). In rats fasted for 48 h and treated with omeprazole the circulating
R. DIMALINE, D. EVANS, A. VARRO AND G. J. DOCKRAY gastrin concentration was 142 + 36 pmol 1', which was significantly greater than that in either fed ad libitum or fasted vehicle-treated rats (P < 001).
488
Tissue gastrin The concentrations of immunoreactive gastrin in the antral tissue of rats fed ad libitum measured with antibody L2 were 1P25 + 017 nmol g-i, and these were reduced
I -
E E
'a
:t
20 40 60 80 100 120140 Elution volume (ml)
Fig. 4. Separation by DEAE ion exchange chromatography of different forms of progastrin-derived peptides measured with antibody L304. In both fasted (A) and fastedomeprazole treated rats (B) four peaks were consistently separated. These correspond to progressively more acidic peptides and in order of elution are (a) the unmodified peptide (CFP-I), (b) phosphorylated (CFP-II), (c) sulphated (CFP-III) and (d) both phosphorylated and sulphated peptides (CFP-IV). See text for estimates of the proportion of progastrin-derived peptides in the phosphorylated state.
by about 50% (0-67 + 011 nmol g-1) in rats fasted for 48 h. In fasted rats treated with omeprazole, however, tissue concentrations of gastrin measured with L2 were not significantly different from those in rats fed ad libitum (099 + 020 nmol g'). Assays using antibody L304 which is specific for the C-terminus of progastrin recorded rather higher concentrations of immunoreactivity compared with antibody L2, in all three groups of rats (Table 2). The same pattern was observed, however, in that in fasted rats there were significantly decreased tissue conoentrations compared with rats fed ad libitum, while in rats treated with omeprazole concentrations were not significantly reduced.
G-CELL CONTROL MECHANISMS Lane
489
7 8 9 10 11
1 2 3 4 5 6
A 28 s18 s--
Lane
1 2 3 4 5 6
7 8 9 10 1112
B 28 s_ 18 s -_-
Fig. 5. A, northern blot analysis of gastrin mRNA levels in 20 ,ug samples of total antral RNA from rats fasted for 48 h (lanes 1-6) or fed ad libitum (lanes 7-11). Arrows indicate the positions of ribosomal subunits. B, Northern blot analysis of gastrin mRNA levels in 20 jug samples of total antral mRNA from rats fasted for 48 h (lanes 1-6) or fasted for 48 h with omeprazole treatment (7-12). TABLE 2. Tissue and plasma gastrin concentrations in fed and fasted rats (mean ± s.E.M., n=
Plasma gastrin
Group Fed ad libitum Fasted, vehicle Fasted, omeprazole
(pmol l-1) 50 0+4-8
6)*
Tissue gastrin (L2) Tissue gastrin (L304) (nmol g-1) (nmol g-1)
1P25+0417
1P75+0 25
14-4 + 4-3t 0.67 + 011 t 1P06 ± 041I t 142 + 36t 0-99+0-20 1P31+ 0419 * Plasma gastrin was measured with antibody L2 specific for the C-terminal of G17. Tissue gastrin concentration was measured with this antibody and with antibody L304 which is specific for the C-terminal fragment of rat progastrin. t Significantly different from rats fed ad libitum (P < 0 05)
The C-terminal fragments of progastrin that react with antibody L304 occur in several different forms that can be separated by ion exchange chromatography. In order of elution from DEAE these are unmodified, phosphorylated, sulphated, and both phosphorylated and sulphated (Fig. 4). Fasting decreased the proportion of progastrin-derived peptides that occurs in the phosphorylated state; thus in rats fed ad libitum, 51-6 + 4-4% of progastrin-derived C-terminal flanking peptide was phosphorylated compared with 33-0+444% in rats which were fasted and treated with vehicle (P < 0 05). In fasted rats treated with omeprazole the phosphorylation of progastrin-derived peptides (52-2 + 2-9 %) was not significantly different from that in rats fed ad libitum. The proportion of progastrin-derived C-terminal flanking peptide that was sulphated (approximately 90%) was not significantly changed by fasting.
490
R. DIMALINE, D. EVANS, A. VARRO AND G. J. DOCKRAY
mRNA Northern blots of gastrin mRNA revealed a single band in both fasted and fed rats that was compatible with an mRNA species of 650 bases. The quantification of mRNA by scanning laser densitometry indicated that the abundance of gastrin mRNA in fasted rats was 65-6 + 107 % of that in rats fed ad libitum (P < 005) (Fig. 5). In contrast, in fasted rats treated with omeprazole gastrin mRNA was 134'3+22-1 % of that in rats fed ad libitum (Fig. 5). DISCUSSION
The decrease in tissue and plasma gastrin concentrations that occurs on withdrawal of food in the rat has been described by several groups (Lichtenberger et al. 1975; Track et al. 1978; Mortensen et al. 1979), and is confirmed in the present study. In addition, in fasting there is (a) a depression of tissue gastrin mRNA levels which suggests either depressed gastrin gene transcription or increased mRNA degradation, and (b) a decrease in the proportion of progastrin-derived peptides that occur in the phosphorylated state, which is a marker for progastrin processing. Together the data indicate that in fasted rats, both gastrin release and biosynthesis are reduced. The primary observation of the present study is that omeprazole reverses these changes in G-cell function. The major luminal factors that control pyloric antral G-cells are protein in the gastric lumen (which stimulates) and acid (which inhibits). The H+-K+-ATPase inhibitor omeprazole is a powerful inhibitor of acid secretion. The dose we used was shown to produce achlorhydria for at least 24 h. It is known that in rats fed ad libitum similar doses of omeprazole produce hypergastrinaemia, increased gastrin mRNA and eventually G-cell hyperplasia (Allen, Bishop, Daly, Larsson, Carlsson, Polak & Bloom 1986; Larsson, Carlsson, Mattsson, Lundell, Sundler, Sundell, Wallmark, Watanabe & Hakanson, 1986; Brand & Stone, 1988). The demonstration that omeprazole reverses the inhibitory effects of fasting on the G-cell suggests that the latter are attributable to the unrestrained inhibition of G-cell function by luminal acid. It may be that the rat is particularly susceptible to the inhibitory effects of acid in the fasting state because basal rates of secretion are relatively high. Thus when normalized to body weight, basal acid secretion in the rat is 400-600 #smol kg-' h-'; and is 20-30 % of maximal acid output, whereas in dog and man, basal acid output is lower whether expressed relative to body weight (5-40 #nmol kg-' h-') or as a proportion of maximal, i.e. 1-10 % (Debas, 1987). Rates of acid secretion after 48 h fasting in rat were similar to those at 24 h, and to those found after an overnight fast (approximately 15 h). Since plasma gastrin decreased sharply with 48 h fasting it would appear that it is not responsible for maintaining basal acid secretion. There is now good evidence that acid releases somatostatin from pyloric antral D-cells which in turn acts as a paracrine inhibitor of gastrin release and gastrin synthesis (Saffouri, Weir, Bitar & Makhlouf, 1980; Short, Doyle & Wolfe, 1984; Brand & Stone, 1988; Dockray & Gregory, 1989; Karnik, Monahan & Wolfe, 1989). It seems possible, then, that in the absence of stimulation by food basal acid secretion in fasted rats releases somatostatin which acts to depress gastrin synthesis
G-CELL CONTROL MECHANISMS 491 and release. These data imply that there is tonic inhibition of the rat G-cell by acid. Previous studies in other species (dog, man) have generally failed to demonstrate that acute alkalinization of the empty stomach increases plasma gastrin (Walsh & Grossman, 1975). Whether or not maintained alkalinization of the empty stomach for 48 h, as in this study, influences human or dog G-cell function remains to be seen. For reasons mentioned above, the rat may be the species of choice for demonstrating the tonic inhibitory effects of gastric acid on G-cells. Protein or peptide synthesis is in principle controlled at several levels including mRNA transcription, mRNA degradation, translational rates and post-translational processing. The data on gastrin mRNA levels provide clear evidence that either transcription is depressed or degradation increased with fasting; in the dog there is evidence that somatostatin affects both (Karnik et al. 1989). Antibody L2 reacts with biologically active C-terminally amidated gastrin, and would not reveal inactive precursors of intermediates; on its own therefore assays with this antibody cannot provide much insight into the control of post-translational processing. However, antibody L304 reacts both with progastrin and its C-terminal fragment which means it can detect the products of gastrin mRNA translation, regardless of subsequent processing. In many species there is a 1:1 stoichiometry between amidated gastrin and progastrin C-terminal fragments (Dockray & Gregory, 1989). In the rat, however, there are appreciable quantities of a Gly-extended .biosynthetic intermediate, which is the immediate precursor of the amidated gastrins (Daugherty & Yamada, 1989). The lower tissue concentrations measured with L2 compared with L304 can be accounted for by the failure to convert fully Gly-extended intermediates to G17. Assays with antibody L304 showed a depression in tissue concentrations of immunoreactivity comparable to those with L2, which indicates depressed production of progastrin, and so allows us to exclude the possibility that the low tissue and plasma gastrin in fasted rats is attributable to a failure to convert inactive precursor forms to biologically active amidated gastrins. The C-terminal fragment of progastrin is phosphorylated in many species (Dockray et al. 1987; Desmond, Varro, Young, Gregory, Nemeth & Dockray, 1989), and there is evidence that depressed phosphorylation is associated with decreased synthesis (Varro et al. 1990b). The finding of reduced phosphorylation of progastrin-derived peptides in fasted rats contributes to the idea that phosphorylation provides an index of gastrin biosynthetic activity. More generally, however, the reversal of phosphorylation in omeprazole-treated rats is direct evidence that at least some of the post-translational processing events in gastrin biosynthesis are regulated by the gastric luminal contents. It is well recognized that prolonged achlorhydria in ad libitum fed animals leads to G-cell hyperplasia, hypergastrinaemia, and increased tissue gastrin mRNA (see Dockray & Gregory, 1989). Our data indicate that in the rat, inhibition of acid alone is sufficient to produce the same effect, i.e. direct stimulation by food is not necessary for maintenance of plasma gastrin or of gastrin synthesis rates. In the present experiment we did not estimate G-cell numbers, but since these cells turn over slowly we assume the G-cell population did not change (Thompson, Price & Wright, 1990). The data strongly suggest that luminal acid is able to work acutely at three levels to control G-cell function, namely by regulating progastrin mRNA abundance,
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progastrin post-translational processing and gastrin secretion. Since plasma gastrin concentrations in omeprazole-treated rats exceeded those in fed ad libitum rats it would appear that depression by acid is actually more important than stimulation by food for controlling the secretory activity of rat G-cells. We are grateful to the MRC for financial support, to Christine Carter for help in preparing the manuscript, and to Dr D. Edgar for the use of a scanning laser densitometer. REFERENCES
ALLEN, J. M., BISHOP, A. E., DALY, M. J., LARSSON, H., CARLSSON, E., POLAK, J. M. & BLOOM, S. R. (1986). Effect of inhibition of acid secretion on the regulatory peptides in the rat stomach. Gastroenterology 90, 970-977. BRAND, S. J. & STONE, D. (1988). Reciprocal regulation of antral gastrin and somatostatin gene expression in omeprazole-induced achlorhydria. Journal of Clinical Investigation 82, 1059-1066. CHIRGWIN, J. J., PRZBYLA, A. E., MACDONALD, R. J. & RUTTER, W. J. (1979). Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18, 5294-5299.
DAUGHERTY, D. & YAMADA, T. (1989). Posttranslational processing of gastrin. Physiological Reviews 69, 482-502. DEBAS, H. T. (1987). Peripheral regulation of gastric acid secretion. In Physiology of the Gastrointestinal Tract, 2nd edn, ed. JOHNSON, L. R., pp. 931-945. Raven Press, New York. DESMOND, H., PAUWELS, S., VARRO, A., GREGORY, H., YOUNG, J. & DOCKRAY, G. J. (1987). Isolation and characterization of the intact gastrin precursor from a gastrinoma. FEBS Letters
210, 185-188. DESMOND, H., VARRO, A., YOUNG, J., GREGORY, H., NEMETH, J. & DOCKRAY, G. J. (1989). The constitution and properties of phosphorylated and unphosphorylated C-terminal fragments of progastrin from dog and ferret antrum. Regulatory Peptides 25, 223-233. DIMALINE, R., CARTER, N. & BARNES, S. (1986). Evidence for reflex adrenergic inhibition of acid secretion in the conscious rat. American Journal of Physiology 251, G615-618. DOCKRAY, G. J., BEST, L. & TAYLOR, I. L. (1977). Immunochemical characterization of gastrin in pancreatic islets of normal and genetically obese mice. Journal of Endocrinology 72, 143-151. DOCKRAY, G. J. & GREGORY, R. A. (1989). Gastrin. In Handbook of Physiology - The Gastrointestinal System II, chap. 15, ed. MAKHLOUF, G. M., pp. 311-336. American Physiological Society, Bethesda, MD, USA. DOCKRAY, G. J., VARRO, A., DESMOND, H., YOUNG, J., GREGORY, H. & GREGORY, R. A. (1987). Post-translational processing of the porcine gastrin precursor by phosphorylation of the COOHterminal fragment. Journal of Biological Chemistry 262, 8643-8647. FULLER, P. J., STONE, D. L. & BRAND, S. J. (1987). Molecular cloning and sequencing of a rat preprogastrin complementary deoxyribonucleic acid. Molecular Endocrinology 1, 306-311. KARNIK, P. S., MONAHAN, S. J. & WOLFE, M. (1989). Inhibition of gastrin gene expression by somatostatin. Journal of Clinical Investigation 83, 367-372. LARSSON, H., CARLSSON, E., MATTSSON, H., LUNDELL, L., SUNDLER, F., SUNDELL, G., WALLMARK, B., WATANABE, T. & HAKANSON, R. (1986). Plasma gastrin and gastric enterochromaffinlike cell activation and proliferation: Studies with omeprazole and ranitidine in intact and antrectomized rats. Gastroenterology 90, 391-399. LICHTENBERGER, L. M., LECHAGO, J. & JOHNSON, L. R. (1975). Depression of antral and serum gastrin concentration by food deprivation in the rat. Gastroenterology 68, 1473-1479. MORTENSEN, N. J. MCC., MORRIS, J. F. & OWENS, C. (1979). Gastrin and the ultrastructure of G cells in the fasting rat. Gut 20, 41-50. SAFFOURI, B., WEIR, G. C., BITAR, K. N. & MAKHLOUF, G. M. (1980). Gastrin and somatostatin secretion by perfused rat stomach: Functional linkage of antral peptides. American Journal of
Physiology 238, G495-501. SHORT, G. M., DOYLE, J. W. & WOLFE, M. M. (1984). Effect of antibodies to somatostatin on acid secretion and gastrin release by the isolated perfused rat stomach. Gastroenterology 88, 984-988.
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