Effects of Fasting and Pentagastrin on Protein Synthesis by Isolated Gastric Mucosal Ribosomes in a Cell-Free System

Vol. 73, No.5

GASTROENTEROLOGY 73:1060-1064, 1977 Copyright © 1977 by the American Gastroenterological Association

Printed in U.S A.

EFFECTS OF FASTING AND PENTAGASTRIN ON PROTEIN SYNTHESIS BY ISOLATED GASTRIC MUCOSAL RIBOSOMES IN A CELL-FREE SYSTEM ADHIP P . NANDI MAJUMDAR, PH.D., AND NILS GOLTERMANN, M.D. Institute of Medical Biochemistry, University of Aarhus, Aarhus C, Denmark

Groups of rats were either fed ad libitum or fasted for 48 hr, and pentagastrin (500 J.tg per kg) was then injected to one fasted group, while the other fasted and fed groups received 0.9% saline. The capacity of isolated gastric mucosal polyribosomes to synthesize protein in a cell-free system was studied. Liver cell sap, prepared from normal fed rats was used as a source for activating enzymes. The rate of [14 C]amino acid incorporation into protein by gastric mucosal polyribosomes from nonfasted and pentagastrininjected rats was found to be considerably higher than that of the fasted control. A study of the time interrelationship of protein synthesis after a dose of either pentagastrin or saline revealed that 1 hr after the hormone injection ribosomal protein synthesis was 40% higher than in the control. But 1.5 and 6 hr after the hormone treatment the increments were 22 and 15% above the respective controls. A prolongation of the fasting period itselffrom 48 to 54 hr caused a slight 7% reduction in ribosomal protein synthesis. Protein to phenylalanine ratio, which represents a ratio of polysomes to monosomes, was found 100% above the fasted controls 1 hr after pentagastrin injection. It was further observed that whereab :in the absence ofpoly(U) the incorporation of [14 C]phenylalanine into protein by polyribosomes from the pentagastrin-injected rats was 65% higher than the fasted control, in the presence of poly(U), the control ribosomes showed a 20% increased incorporation. Polyphenylalanine synthesis by the control ribosomes was also found to be 30% above the hormone-treated group. The more active response of the control ribosomes to poly(U) is thought to be attributable to the presence of a higher proportion of monosomes in the preparation. Overproduction of gastrin produces gastric mucosal hyperplasia, 1· 2 whereas a lack of this hormone after gastrectomy or antrectomy results in atrophy of parts of the digestive tract. 3• 4 Moreover, it has been demonstrated that chronic as well as a single injection of pentagastrin but not histamine stimulate protein, RNA, and DNA synthesis in gastric and duodenal mucosa.4--11 Enhanced amino acid incorporation into protein of gastric and duodenal mucosa has also been observed after administration ofhuman synthetic gastrin G-17 in rats. 9 These and other observations led Johnson to hypothesize that gastrin is a trophic hormone for parts of the gastrointestinal tract. 12 Earlier studies of the effect of gastrin on protein synthesis have measured amino acid incorporation into protein either in vivo or in vitro using tissue homogeReceived February 14, 1977. Accepted May 23, 1977. Address requests for reprints to: Dr. A. P . Nandi Majumdar, Institute of Medical Biochemistry, University of Aarhus, DK-8000 Aarhus C, Denmark. This study was supported by grants from Novo's Fond, Fonden til Laegevidenskabens Fremme, and the Laegevidenskabelige Forskningsrlanad, Denmark. The authors are grateful to Miss Karin Morre and Mrs. Lene Pedersen for their excellent technical assistance. They wish to thank Professor Jens Rehfeld for his interest and valuable criticism.

nates. 8 • 9 Although the experiments reported so far clearly demonstrate that gastrin stimulates amino acid incorporation into proteins, the biochemical changes associated with the hormone-stimulated protein synthesis in the gut have not been studied in detail. In the present investigation gastric mucosal polyribosomes, isolated from fasted, fed, and pentagastrintreated-fasted rats were studied for their ability to support protein synthesis in a cell-free system.

Materials and Methods Adult Wistar rats of both sexes, weighing between 175 and 200 g were used in this study. The rats were either maintained on ad libitum commercial laboratory diet or fasted for 48 hr before the experiment. All animals had access to water throughout. During fasting the animals were housed in wirebottomed cages to prevent coprophagy. In the present investigation pentagastrin (Peptavalon, Imperial Chemical Industries, Macclesfield, England) was administered at a dose of 500 J.Lg per kg. This dose was chosen because it was shown by Johnson et al. 8 to produce maximum stimulation in [ 14 C]leucine incorporation into protein of the gastric mucosa in vitro. The fasted rats were injected intraperitoneally with either pentagastrin or an equivalent volume of 0.9% saline, and killed at different intervals. The nonfasted rats received 0.9% saline the same way and were killed 1 hr later. The stomach was quickly excised and opened, and the

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PENTAGASTRIN-INDUCED PROTEIN SYNTHESIS

contents were rinsed out with cold 0.9% saline. The mucosal layer was lightly scraped out with a cold spatula, and the scrapings were collected in a centrifuge tube containing 0.25 M sucrose-1 mM MgCl2 . The scrapings were recovered by low speed centrifugation and were stored at -90aC for further use. Gastric mucosal scrapings from 3 to 5 rats were pooled to obtain sufficient material for isolation of ribosomes. Polyribosomes were isolated from postnuclear supernatant fluid, essentially according to the procedure described by Venkatesan and Steele.' 3 Briefly, the gastric mucosal scrapings were homogenized in 8 volumes of cold homogenizing buffer containing 0.25 M sucrose, 0.05 M Tris-HCl, pH 7.4, 0.025 M KCl, and 0.005 M MgCl 2 in a glass-Teflon homogenizer. Polyvinyl sulfate (50 JLg per ml) was added before homogenization as ribonuclease inhibitor. Homogenate was treated with a final concentration of 1% Triton X-100, and centrifuged without delay at 1417 x g for 5 min to remove nuclei. The postnuclear fractions were then made 1.3% with respect to sodium deoxycholate. Aliquots of 5 ml were layered over 3.4 ml of 1.38 M sucrose in the homogenizing buffer, and were centrifuged at 226,000 x g for 4 hr in a Beckman L5-50 ultracentrifuge (Beckman Instruments). The ribosomal pellet was then suspended in TKMEDG buffer (0.05 M Tris-HCl, pH 7.4, 0.025 M KCl, 0.005 M MgCl 2 , 0.001 M dithiothreitol (DTT), 0.00025 M ethylenediaminetetraacetate (EDTA), and 25% glycerol). The A2so nm:A2so nm ratio was generally between 1. 70 and 1. 75. The concentration of ribosomal RNA was determined as described previously. 14 Cell sap was prepared from normal rat livers by homogenizing tissues in 2.5 volumes of cold TKM buffer (0.05 MTris-HCl, pH 7.4, 0.025 M KCl, 0.005 M MgCl 2 ) containing 0.25 M sucrose, as described earlier. 15 Cell sap was passed through a Sephadex G-25 column, previously equilibrated with sucrose-TKM buffer, to remove low molecular weight materials. Protein concentration of cell sap was measured by the method of Lowry et al. 16 Conditions for endogenous messenger RNA (mRNA)-directed amino acid incorporation were essentially the same as described earlier. 17 Unless otherwise stated, the standard incubation medium contained in a final volume of 0.125 ml:6.25 f.lillOles ofTris-HCl, pH 7.6; 12.5 JLmoles ofKCl; 1.1 JLmoles of Mg acetate; 0. 75 JLmoles of 2-mercaptoethanol; 0.125 JLmoles of ATP; 0.025 f.Lmole of GTP; 0.615 JLmole of creatine phosphate; 6.25 f.Lg of creatine phosphokinase; 1.0 mg of cell sap-protein, ribosomal suspensions (15 to 18 f.Lg of RNA), and 0.0625 JLCi of "C-labeled protein hydrolysate (54 mCi per matom carbon, Radiochemical Centre, Amersham, England). Incubations were carried out under aerobic conditions at 37°C for 30 min or for time periods described in the legends to figures and tables. Incubations were terminated by the addition of 2 ml of 7% trichloroacetic acid. The samples were left on ice for 30 min, heated for 20 min at 90°C , and cooled for 20 min. The protein precipitates were collected on Whatman glass fiber filters (GF/ C) and washed exhaustively with 5% trichloroacetic acid, three times with ether-ethanol (1:1, v/v), and once with ether alone. To each filter 0.5 ml of Soluene-100-isopropanol (1:1, v/ v) was added, and after 1 hr at room temperature, 50 JLl of concentrated formic acid were added to each. The radioactivity was counted in 10 ml oflnsta-fluor (Packard Instrument Company, Downers Grove, Ill.) in a LKB-Wallac scintillation spectrometer (LKB Instruments, Inc., Rockville, Md.) after temperature equilibration.

Results and Discussion In the present investigation gastric mucosal polyribosomes were isolated from six groups of 48-hr-fasted rats,

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three nonfasted groups, and six pentagastrin-injected groups. Thus a total of 15 different ribosomal preparations were utilized. The ability of isolated polyribosomes to synthesize protein in a cell-free system was then investigated. Liver cell sap (105,000 x g supernatant), prepared from normal fed rats was used as a source for activating enzymes. Gastric mucosal polyribosomes, prepared directly from Triton X-100 treated postnuclear supernatant were found to be active in incorporating [14 C]amino acid into protein in a cell-free system. Polyribosomes isolated by the present technique were also considered to be sufficiently pure, in that the absorbance ratios, 260:280 and 260:235, were 1. 70 to 1. 75 and 1.45 .to 1.50, respectively. These values are in good agreement with those reported for isolated ribosomes from other tissues. 13 • 18• 19 In the first series of experiments, the rate of incorporation of [14 C]amino acid into protein by gastric mucosal polyribosomes was studied over a period of 60 min. Assay of ribosomal activity in vitro showed that 14 [ C]amino acid was incorporated very rapidly for the first 8 to 10 min; thereafter the rate declined until after 30 min essentially no further synthesis occurred (fig. 1). The results of the time course study also revealed that the incorporation of [! 4 C]amino acid into protein by both nonfasted and the pentagastrin-injected group was considerably higher as compared to fasted controls (fig. 1). The results presented in figure 1B showed that ribosomes from the hormone-treated group had higher protein synthesizing activity than the nonfasted rats, whereas figure 1A revealed an opposite picture. In this connection it should be mentioned that in the present investigation, where three groups of nonfasted rats

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FIG. 1. Time course incorporation of 14 C-labeled amino acid into protein by gastric mucosal polyribosomes from fasted (0-- -0), nonfasted (6--6), and pentagastrin-treated (x--x) rats. Rats were killed 1 hr after saline or pentagastrin (500 !Lg per kg) injection. In each group tissues from 5 rats were pooled for isolation of ribosomes. The results of two experiments are shown inA and B. Each point on the curve represents an average of triplicate determinations. Zerotime values were subtracted from the experimental point. In experiment A the results were obtained from one ribosomal preparation from each group, whereas the results of experimentB were from two separate preparations from each group.

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MAJUMDAR AND GOLTERMANN

were used, gastric mucosal polyribosomes from one of the groups showed increased capacity to synthesize protein in vitro (fig. 1A and table 1). Furthermore, it should also be noted that we have occasionally observed such a phenomenon in the course of other similar studies. These inconsistent findings of increased protein synthesis by ribosomes from nonfasted rats compared to the pentagastrin-injected group are probably attributable to the amount of food consumed by the nonfasted rats before sacrifice. To ascertain whether the present in vitro system effectively measures ribosomal protein synthesis the sensitivity of the cell-free system to various inhibitors of protein synthesis was investigated. Addition of puromycin and cycloheximide, and omission of GTP- and ATPgenerating system reduced [I 4 C]amino acid incorporation to 4 to 30% of the complete system (table 1). Polyribosomes from all three groups showed similar sensitivity to either addition of protein synthesis inhibitors or omission of ATP and GTP. Because ionic environment is known to affect protein synthesis, the optimum requirements for Mg2 + and K+ were determined in the next experiment. Gastric mucosal polyribosomes from fasted, nonfasted, and pentagastrin-treated rats had an absolute requirement for Mg2+. A sharp peak of incorporation was found to occur with a concentration of 8.8 mM (fig. 2). This value is somewhat higher than what has been reported for cellfree protein synthesis by ribosomes from other tissues.1s-zo In the case of K+, maximal rate of protein synthesis by gastric mucosal polyribosomes was observed over a broad range of concentration (75 to 90 mM), and was the same for all three groups (fig. 2). In the next experiment the time interrelationship of ribosomal protein synthesis after a dose of pentagastrin was studied. Six groups of 48-hr fasted rats were killed at various intervals after a single injection of either pentagastrin or saline, and the capacity of isolated gas-

Vol. 73,No.5

tric mucosal polyribosomes to synthesize protein in a cell-free system was measured. One hour after the hormone treatment ribosomal capacity to synthesize protein was 40% higher than the saline-treated control (fig. 3). The stimulatory effect of pentagastrin declined rapidly with time; 1.5 and 6 hr after the hormone injection the values were 25 and 15% higher, respectively, compared to the corresponding control groups. The ribosomal protein synthesis by 1.5-hr control group was essentially the same as that of the 1-hr saline control.

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1. Effects of various inhibitors on the incorporation of "Clabeled amino acid into protein by gastric mucosal polyribosomes from fasted , nonfasted, and pentagastrin-treated fasted rats• TABLE

Radioactivity incorporated Incubation condition Fasted

Complete system Without GTP- and ATP-generating system Cycloheximide (1.5 mM) Puromycin (1 mM)

1061 (100) 128 (12) 318 (30) 204 (19)

Fasted + penFed tagastrin counts per min

1565 (100) 66 (4) 337 (21) 312 (20)

1478 (100) 81 (6) 416 (28) 267 (18)

a Rats were killed 1 hr after saline or pentagastrin (500 p..g per kg) treatment. Incuabtions at 37°C for 30 min were carried out as described in Materials and Methods. Each value is an average of duplicate determinations. Zero-time values (50 to 70 counts per min) were subtracted from the experimental point. Figures in the parentheses represent percentage of incorporation compared to respective complete system. Complete incubation systems without ribosomes had 110-125 cpm.

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FIG. 3. Time-dependent response in protein synthesis by gastric mucosal ribosomes after a dose of pentagastrin or saline. Groups of 48-hr fasted rats were injected with either saline or pentagastrin (500 p..g per kg) and killed 1, 1.5, and 6 hr afterward. In each group tissues from 4 rats were pooled. Incubations were carried out at 37°C for 30 min. Zero-time values were subtracted from the experimental point. The results are expressed as means± SEM of five to eight determinations. The differences between the pentagastrin-injected and control values were statistically analyzed by using student's t-test.

November 1977 TABLE

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PENTAGASTRIN-INDUCED PROTEIN SYNTHESIS

2. ['"'C]Phenylalanine incorporation into protein by gastric mucosal polyribosomes in the presence and absence of poly(U) a Incorporation

Treatment Minus poly(U)

Saline-treated control Pentagastrin-treated

Plus poly(U) Polyphenylalanine' I0 - 3 x counts per min/mg rRNA

34.2 ± 1.1 (100) 56.3 ± 1.0c (165)

586.8 ± 52.3 (100) 481.3 ± 32.3 (82)

552.6 (100) 425.0 (77)

Protein/phenylalanine ratio'

0.06 (100) 0.12 (200)

a Two groups of 48-hr fasted rats (3 rats in each group) were killed 1 hr after saline or pentagastrin (500 1-
However, in the 6-hr control group there was a slight 7 to 10% reduction in protein synthesis when compared with the initial control groups (fig. 3). This could be interpreted as owing to prolongation of the starvation period. Thus when the ribosomal protein synthesis after 6 hr of pentagastrin treatment was compared with the initial1-hr saline control only a 7% stimulation could be observed. Factors which may limit the rate of protein synthesis in a cell-free system include the proportion of ribosomes in the preparation bound to mRNA, inasmuch as the monomers and dimers are largely inactive in endogenous mRNA-directed protein synthesis. 21 To ascertain whether gastric mucosal ribosomes, isolated from pentagastrin-treated rats, would contain a higher proportion of polysomes compared to the saline-treated fasted control, the ratio of polysomes to monosomes in both preparations was determined by cell-free protein synthesis in the presence and absence of poly(U). 14 [ C)phenylalanine was used as a tracer. It is believed that in the absence of poly(U), [14 C]phenylalanine would be incorporated into protein which is synthesized by existing polysomes in the preparation. 22• 23 In the presence of poly(U) however, the incorporation of 14 [ C)phenylalanine can be attributed to the formation of polysomal structure formed by regrouping of monosomes. Therefore the ratio between the synthesis of protein and polyphenylalanine would represent a ratio of polysomes to monosomes. The results of such an experiment carried out with gastric mucosal ribosomes from fasted and pentagastrin-treated rats revealed a 100% higher protein to phenylalanine ratio for ribosomes prepared from pentagastrin-injected rats as compared to the saline-treated fasted control (table 2) . It was also observed that in the absence of poly(U) the incorporation of [14 C]phenylalanine into protein by ribosomes from the hormone-treated group was 65% higher than the control (table 2). This finding is similar to that observed with [14 C]protein hydrolysate (figs. 1 and 2), confirming further that a single injection of pentagastrin stimulates the capacity of gastric mucosal polyribosomes to synthesize endogenous mRNA-directed protein in a cell-free system. However, in the presence of poly(U), gastric mucosal ribosomes from the saline-in-

jected group showed a 22% higher [! 4 C]phenylalanine incorporation when compared with the pentagastrininjected group (table 2). Furthermore, polyphenylalanine synthesis by the saline-treated control ribosomes was also 30% above the hormone-treated group. The more active response of the control ribosomes to synthetic mRNA poly(U) indicates a deficiency of endogenous mRNA leading to disaggregation of polysomes to more inactive monosomes. The finding of a lowered protein to phenylalanine ratio (represents polysomes to monosomes ratio) in the 48-hr fasted control as compared to the pentagastrin-treated group also indicates the presence of a higher proportion of monosomes in the control preparation. The increased polysomes to monosomes ratio, observed after 1 hr of pentagastrin injection suggests that the hormone stimulates the proportion of mRNA-bound ribosomes in the gastric mucosal cell. This may be attributable to the availability of more mRNA in the cytoplasm for polysome formation after pentagastrin injection. In this connection it may be mentioned that pentagastrin has earlier been shown to stimulate RNA synthesis (probably mRNA) in the gastric mucosa of both antrectomized and fasted rats. 4 • 11 REFERENCES 1. Gregory RA, Grossman MI, Tracy HJ, et al: Nature of the gastric secretagogue in Zollinger-Ellison tumor. Lancet 2:543-544 , 1967 2. Ellison EH, Wilson SD: Further observations on factors influencing the symptomatology manifested by patients with Zollinger-Ellison syndrome. In Gastric Secretion. Edited by TK Shnitka, JAL Gilbert, RC Harrison. New York, Pergamon, 1967, p 363-369 3. Lees F, Grandjean LC: The gastric and jejunal mucosa in healthy patients with partial gastrectomy. Arch Intern Med 101:94379451, 1968 4. Johnson LR, Chandler AM: RNA and DNA of gastric and duodenal mucosa in antrectomized and gastrin-treated rats. Am J Physiol 224:937-940, 1973 5. Crean GP, Marshall MW, Rumsey RDE: Parietal cell hyperplasia induced by the administration of pentagastrin (ICI 50,123) to rats. Gastroenterology 57:147-155, 1969 6. Stanley MD, Coalson RE , Grossman MI, et al: Influence of secretin and pentagastrin on acid secretion and parietal cell numbers in rats. Gastroenterology 63:264-269, 1972 7. Hansen OH, Pedersen T, Larsen JK, et al: Effect of gastrin on gastric mucosal cell proliferation in man. Gut 17:536-541, 1976

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8. Johnson LR, Aures D, Yuen L: Pentagastrin-induced stimulation of protein synthesis in the gastrointestinal tract. Am J Physiol 217:251-254, 1969 9. Johnson LR, Aures D, Hakanson R: Effect of gastrin on in vivo incorporation of 14 C-leucine into protein of the digestive tract. Proc Soc Exp Bioi Med 132:996-998, 1969 10. Enochs MR, Johnson LR: Pentagastrin stimulates tissue growth in stomach and duodenal tissues by stimulating protein and nucleic acid synthesis. Fed Proc 33:309, 1974 11. Chandler AM, Johnson LR: Pentagastrin-stimulated incorporation of 14 C-orotic acid into RNA of gastric and duodenal mucosa. Proc Soc Exp Bioi Med 141:110-113, 1972 12. Johnson LR: The trophic action of gastrointestinal hormones. Gastroenterology 70:278-288, 1976 13. Venkatesan'N, Steele WJ: Isolation of ribosomes from postnuclear fraction of rat liver in nearly quantitative yield. Biochim Biophys Acta 277:646-650, 1972 14. Trachewsky D, Majumdar APN, Congote LF: Effects of aldosterone and other steroids in vivo on rat kidney cortex. Alteration in translation by isolated ribosomes and in the thiol content of nuclear proteins. Eur J Biochem 26:543-552, 1972 15. Majumdar APN: Iron-loading: effect on the ~ctivity of liver ribosomes in protein synthesis in rats. Nutr Rep Int 13:9-16, 1976 16 .. Lowry OH, Rosebrough NJ, Farr AL, et al: Protein measure-

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