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Nucleic acid metabolism in the gastrointestinal tract of the mouse during fasting and restraint-stress
EXPERIMENTAL
AND
Nucleic
Acid
MOLECULAR
Metabolism
Mouse
during R.
ANTHONY
Department
of Medicine, For
(1968)
PATHOLOGY9,3%-348
IMONDI, Cornell
Cancer
in the Fasting M. University Research, Received
Gastrointestinal and
EARL
BALIS, Medical
New June
York,
Tract
of the
Restraint-Stress’ AND College New
MARTIN
LIPKIN
and Sloan-Kettering York
Institute
10021
21, 1968
The mucosal lining of the mammalian gastrointestinal tract undergoes profound changes during starvation and stress. Decreases in the number of cells lining the crypts and villi of rats starved for 5 days (Hooper and Blair, 1958) and in DNA synthesis in the intestinal mucosa of starved mice (Brown et al., 1963) have been reported. Munro and Goldberg (1964) reported that the feeding of protein-free diets to rats results in a decreased uptake of 3zP into DNA of intestinal mucosa but, had little effect on RNA metabolism. Subjecting animals or humans to stress such as restraint (Rossi et al., 1956)) operative procedures (Penner and Bernheim, 1939), or trauma (Griffith, 1922) produces acute erosions in the upper esophageal, gastric, and duodenal mucosa. Restraintstress has become a common method of producing gastric erosions experimentally. Recently, studies employing this technique have indicated that an inhibition of gastric epithelial cell proilferation precedes the development of erosions (Kim et al., 1967; Lahtiharju and Rytomaa, 1967). These studies have suggested that this inhibition of DNA synthesis and mitosis, with resulting failure to replace extruded epithelial cells, contributes to the development of the gastric erosions. The possible implication of altered nucleic acid metabolism in the development of the stress-induced gastric erosions has prompted an investigation of RNA and DNA metabolism in the gastrointestinal tract of mice subjected to stress. The present communication is a report of such studies and demonstrates differences in the uptake of nucleic acid precursors by cells in various regions of the gastrointestinal tract as a result of fasting and restraint METHODS Male CFW mice weighing 25-27 gm were used in these studies. Four experiments were conducted to measure the incorporation of thymidinemethy13H (TdR-3H; 16.5 c/m&!) into DNA after 16 hours (experiment 1) and 40 hours (experiment 2) and theincorporationof uridine-5-3H (URJH; 16.2 c/mM) into RNA after 16 hours (experiment 3) and 40 hours (experiment 4) of fasting and restraint. Both nucleosides were obtained from New England Nuclear, Boston. In each experiment four normal, four fasted, and four restrained animals were used. The normal mice were 1 This CA-08748;
work was supported by National Institutes I-F2-CA-37, 131.01; and K3-AM-4468. 339
of Health
Grants
and Awards
CA-08921
;
340
IMONDI,
BALIS,
AND
LIPBIN
kept in mouse cages with wood shavings until time of injection, and then transferred to wire-bottomed cages without food or water during the treatment period. Restrained mice were secured in wire mesh screen (Brodie and Hanson, 1960), also without food or water. At the end of each treatment period, each mouse was injected intraperitoneally with either 20 PC TdR-3H (experiments 1 and 2) or with 10 PC UR3H (experiments 3 and 4) and then returned to treatment. After one hour the mice were killed by cervical dislocation. The glandular stomach, duodenum (first 2.5 cm of small intestine), 2.5 cm of mid-jejunum, 2.5 cm of terminal ileum, and 2.5 cm of proximal colon were removed and homogenized with a Teflon pestle in 3 ml of icecold .Ol M Tris buffer (pH 7.5). The acid-insoluble material was precipitated by adding 1 ml of cold 2 N perchloric acid (PCA) and was then washed twice with 2 ml of cold .5 N PCA, once each with 2 ml of 75 %, 95 70, and 100 % ethanol, a 1: 1 mixture of ethanol-ether, and ether. UR-3H-containing material was hydrolyzed in 1 ml of .3 N NaOH at 37°C for 18 hours (Schmidt and Thannhauser, 1945). After precipitating DNA and protein with PCA, an aliquot of the RNA hydrolysate was assayed for RNA by a modification of the Mejbaum (1939) orcinol method. An appropriately diluted sample of RNA nucleotides was mixed with a concentrated HCl solution containing 0.5 % FeC& and 1% orcinol and was heated for 10 minutes at 100°C. The intensity of the green color was read at 670 rnp. Adenylic acid was used as a standard with appropriate correction. Another aliquot of the RNA hydrolysate was assayed for radioactivity in a Packard Tri-Carb Spectrometer with external standardization. The TdR-3H-containing samples were hydrolyzed in .Fi N PCA at 90°C for 15 minutes and assayed for DNA by the diphenylamine procedure of Seibert (1940) and for radioactivity as described above. Analysis of variance was performed and least significant differences between means were calculated (Steel and Torrie, 1960). The terms significant and highly significant are used throughout the text to denote differences between means at the .05 and .Ol probability levels, respectively. RESULTS DNA
Content of the tissues
The gastrointestinal mucosa of animals fasted for 16 hours and 40 hours revealed no gross abnormalities. In one of the animals restrained for 16 hours (experiment 3), there was blood in the lumen indicative of gastric erosions. All animals restrained for 40 hours had blood in the gastric lumen. DNA Content of the stomach did not decrease significantly after 40 hours of fasting or restraint (Figs. 1 and 2). The duodenum of the fasted and restrained animals did show a significant loss of DNA within 16 hours after treatment. The loss of DNA by the entire small intestine and colon was evident in the restrained mice after 40 hours. Incorporation
of TI~R-~H into DNA
The total amount of TdR-3H incorporated into DNA of the various tissues is presented in Figs. 1 and 2. Fasting for 16 hours had little effect on incorporation of
NUCLEIC
ACID
METABOLISM
DURING
pg DNA /tissue Normal
Fasted
Total TdR-3H /tissue
Restraint
Normal
341
STRESS
Fasted
(dpmx 10e3) Restraint
T 515 STOMACH
823-G DUODENUM
841
.
270 . i
JEJUNUM
462----F-
*
194 . +
ILEUM
740
275
COLON
. + 0
1. DNA Content and total amount tissue after 16 hours of fasting and restraint from normal (p < .05) ; l l , Different from .05). FIG.
0 of thymidine-3H incorporated into DNA of each (mean of 4 mice f standard error). l , Different normal (p < .Ol); +, Different from fasted (p <
TdR-3H, except for a significant decreasein duodenum. Fasting for 40 hours resulted in a decreased incorporation of TdR-3H in the ileum only. In the restrained mice, however, TdR-3H incorporation into DNA was markedly decreasedin all tissues at 16 and 40 hours. Except for the colon at 40 hours, the incorporation of TdR-3H was significantly lower in tissues of restrained mice than in those of both normal and fasted mice. In stomach, jejunum, and ileum the differences are highly significant. The specific activities of DNA (dpm/pg DNA) in the various tissues after 16 hours of treatment are presented in Table I. These data illustrate that the reduction in the incorporation of TdR-3H into DNA was greater than the loss of tissue DNA as a result of restraint. This was especially true in the stomach, where the specific activity was reduced 90% in 16 hours of restraint. Decreasesof 40-75% were observed in the other tissues.An unpublished observation in mice by one of us (A.R.I.) revealed that the incorporation of TdR-3H into DNA was reduced by 40% (p < .05) and 65 % (p < .Ol) in the stomach and jejunum, respectively, after 4 hours of restraint-stress.
342
IMONDI,
Incorporation
BALIS,
AND
LIPKIN
of UR-3H into RNA
Total RNA content was significantly lower in the stomach after 16 hours of restraint. Loss of RNA was not significant in the other tissues after 16 hours of treatment (Fig. 3). Incorporation of URJH was increased in all tissues of the fasted anipg DNA /tissue Normal
Fasted
Total TdR-3H /tissue
Restraint
Normal
Fasted
(dpm x 1O-3) Restraint
515 STOMACH
823
195
.
DUODENUM
. +
217
. JEJUNUM
.. +t 0
ILEUM
COLON
*'
FIG. 2. DNA Content and total amount of thymidine-3H incorporated into DNA of each tissue after 40 hours of fasting and restraint (mean of 4 mice f standard error). l , Different from normal (p < .05) ; 0 0, Different from normal (p < .Ol) ; +, Different from fasted (p < .05); + +, Different from fasted (p < .Ol). TABLE
I
INCORPORATION OF THYMIDINE-3H INTO DNA OF FASTING AND RESTRAINT (dpm/pg Normal
Organ
204 348 347 408 361
Stomach Duodenum Je j unum Ileum Colon D Mean
of 4 mice
f
f f f f f
standard
Fasted 4a 14 36 68 39
201 385 439 428 206 error.
f f f f f
AFTER
16 HOURS
DNA) Restraint
67 60 100 76 25
22 205 223 270 94
f f + + +
6 13 46 13 10
NUCLEIC
ACID
METABOLISM
pg RNA /tissue Normal
Fasted
Restraint
DURING
Total UR-3H/tissue Normal
343
STRESS
Fasted
(dpm x1F3) Restqint
1591 STOMACH 0
956 DUODENUM 0
+ JEJUNUM
. + 601 ILEUM 0
COLON
FIG. 3. RNA Content and total amount of uridine-5-3H tissue after 16 hours of fasting and restraint (mean of 4 mice from normal (p < .05); +, Different from fasted (p < .Ol).
incorporated f standard
into error).
RNA of each 0, Different
mals, and the differences were significant’ and highly significant in jejunum and ileum, respectively. When animals were restrained, however, there were no increases except in ileum. Incorporation of UR-3H into stomach RNA of the 16-hour restrained mice was lowest in the mouse with the gastric erosions. The specific activity of the RNA (Table II) was increased in all organs of the fasted group. A significant difference in the fasted jejunum reflects a very high uptake of UR-3H without a concomitant increase in total RNA. After 40 hours of treatment, the decrease in total stomach RNA in the restrained mice is highly significant compared to the normals and significant compared to the fasted mice (Fig. 4). After 40 hours of restraint, the incorporation of UR-3H was significantly decreased in the stomach but not in any other tissues. Fasting for 40 hours resulted in a slight but not significant increase in the uptake of UR-3H in duodenum and ileum. How-
344
IMONDI,
BALIS,
AND
TABLE
LIPKIN
II
INCORPORATION OF URIDINE-3H INTO RNA AFTER 16 HOURS OF FASTING AND RESTRAINT (dpm/pg RNA) Organ
Normal
Stomach Duodenum Jej unum Ileum Colon
16 16 28 22 19
f f f f f
Fasted 21 29 55 44 22
la 1 10 9 9
0 Mean of 4 mice f standard error. b Different from normal and restraint
f f f f f
Fasted
27 19 30 39 19
Total UR-3H/tissue
Restraint
1086
9 7 3 3 5
Normal
Fasted
(dpmx10-31 Restraint
.. + 0
f f f f f
37.1
1586 STOMACH
1 4 3b 8 1
(p < .05).
pg RNA /tissue Normal
Restraint
. 0
18.7
DUODENUM 0
0
1260 JEJUNUM 0
541 ILEUM 0 1185 COLON 0 FIG. 4. RNA Content and total amount tissue after 40 hours of fasting and restraint from normal (p < .05) ; 0 l , Different from .05).
of uridine-5-3H incorporated (mean of 4 mice f standard normal (p < .Ol) ; +, Different
into RNA of each error). l , Different from fasted (p <
ever, these increases, coupled with the change in total RNA in the tissues, result in specific activities (dpm/pg RNA) much higher in the duodenum and ileum of the fasted mice than those in either the normal or the restrained animals (Table III). The differences were significant and highly significant respectively.
NUCLEIC
ACID
METABOLISM
TABLE
DURING
345
STRESS
III
INCORPORATION OF URIDINFPH INTO RNA AFTER 40 HOURS OF FASTING AND RESTRAINT (dpm/pg RNA) Organ Stomach Duodenum Jej unum Ileum Colon
Normal 24 17 30 35 29
f f f f f
Fasted 4a 1 4 7 4
a Mean of 4 mice f standard error. b Different from normal and restraint c Different from normal and restraint
28 23 44 83 36
f f f f f
Restraint 4 2b 4 6” 6
25 13 32 36 32
f f f f f
5 3 9 7 4
(p < .05). (p < .Ol).
DISCUSSION Fasting and nucleic acid metabolism Fasting for up to 40 hours had no appreciable effect on either the amount or synthesis of DNA in the gastric wall. Only in duodenum did fasting result in decreased synthesis and loss of DNA. The reason for the early decrease in uptake of TdRJH by the fasted ileum is not clear since it was not evident at 40 hours. The RNA content of the gastrointestinal tissues was not affected by fasting. However, the uptake of UR-3H was increased in the small gut after 16 hours of fasting and returned to normal by 40 hours. Clark et al. (1957) reported a decrease in uptake of 32P by liver RNA in rats on the first day of protein-free feeding with an increase thereafter. These changes were attributed to a breakdown of liver RNA during protein depletion resulting in an enlargement of RNA precursor pools. Later, the pools shrink and the injected label is more extensively utilized. This apparent contrast in nucleic acid metabolism between intestine and liver is not unexpected in view of their marked differences in cellular activity. In addition, the rate of cell renewal is very rapid in the intestine but extremely slow in the liver. Stress and nucleic acid metabolism Previous studies using mitotic counts (Rasanen, 1963) and microradioautography (Kim et al., 1967; Lahtiharju and Rytomaa, 1967) have demonstrated that acute and chronic stress decrease the rate of cell division in stomach and epidermis of rodents. The present study confirms these findings and demonstrates that t#he decrease in DNA synthesis occurs throughout the gastrointestinal tract within 16 hours and even as early as 4 hours after the initiation of restraint-stress. Later, the total amount of DNA in duodenum and jejunum also decreased. Since the turnover rate of epithelial cells is somewhat greater in small intestine than in either st’omach or colon (Walker and J‘eblond, 195S), the marked reduction in DNA synthesis coupled with the continued cell loss results in earlier loss of DNA from t’he small intestine than from the other tissues. Metabolism of RNA in the small intestine and colon was less affected by restraint than was that of DNA. This was not true, however, for the stomach, which lost about 50 %. of its total RKA after 16 hours of restraint without decreased incorpora-
346
IMONDI,
BALE,
AND
LIPKIN
tion of UR-3H. Since ribosomal RNA (rRNA) constitutes about 80% of the total RNA and is not labeled to any extent by a l-hour pulse (Balis et al., 1958; Graham and Rake, 1963), it appears reasonable to suggest that most of the RNA lost by the restraint stomach was of ribosomal origin. The incorporation of UR-3H is probably indicative of a continuing synthesis of the rapidly metabolized messenger RNA (mRNA) . The specific mechanisms initiating this alteration in nucleic acid metabolism are not known. Various hormones have been shown to influence nucleic acid synthesis in mammary tissue (Kumaresan and Turner, 1966; Thibodeau and Thayer, 1967), while growth hormone has been shown by Leblond and Carriere (1955) to overcome the decrease in cell proliferation in the intestinal mucosa resulting from hypophysectomy. It has been suggested that adrenal hormones are responsible for decreasing mitotic activity in the epidermis of stressed animals (Bullough, 1952), while corticosteroids cause a greater reduction in mitotic activity in the stomach than in the duodenum (Lahtiharju et al., 1964). Heparin also depresses mitotic activity in the stomach but has no effect on cell proliferation in the small bowel or colon (Rasanen et al., 1966). Experimental lesions in the hypothalamus also cause erosions and hemorrhage in cats (Feldman et al., 1961) and, recent unpublished observations in this laboratory indicate that these result in a depression of cell proliferation in the stomach of rats. Nucleic acid metabolzsm and the development of gastric erosions The marked depression in DNA synthesis, accompanied by a loss of RNA, was followed by the development of gastric erosions. The eventual decrease in synthesis of RNA detected in gastric tissue after 40 hours of stress accompanied the occurrence of erosions in the gastric mucosa. A single animal which manifested erosions after 16 hours of restraint also incorporated less of UR-3H into RNA. These findings suggest that the initial loss of rRNA and the ensuing decrease in the synthesis of mRNA are part of a sequence of events leading to the destruction of epithelial cells of the gastric mucosa. Ribosomes are the site of protein synthesis and it follows that their loss of RNA should result in impaired protein synthesis. Although a relationship between ribosomal function and muco-protein synthesis has not been demonstrated, it is likely that some connection does exist either directly or through the enzymes involved in mucus production. Therefore, loss of rRNA could lead to a decrease in the synthesis of mucus. Indeed, a decrease in gastric mucus has been associated with the appearance of gastric erosions in rats (Hakkinen et al., 1966; Robert et al., 1963) and in mice (Kim et al., 1967). The relationship between RNA and mucus production on one hand and the appearance of erosions on the other is particularly significant in view of the results observed in the small intestine and colon. Here the synthesis of DNA was reduced, but no marked decreases in RNA content or synthesis were noted and erosions were not observed in these regions. It is also known that RNA and protein synthesis are prerequisites for synthesis of DNA (Baserga et al., 1967), which, in turn, is needed for mitosis. Hence, RNA synthesis is important for the maintenance of normal cell replacement, and any’im-
NUCLEIC
ACID
METABOLISM
DURING
347
STRESS
pairment in this process will lead to decreased cell proliferation the tissue to compensate for the loss of cells.
and the inability
of
SUMMARY The uptake of thymidine-methyl-3H and uridine-5-3H into DNA and RNA of gastrointestinal tissues of mice has been measured after 16 and 40 hours of fasting and restraint. Fasting had little effect on DNA or RNA metabolism in the gastrointestinal tract except for increases in the uptake of uridine in the small intestine. These increases are thought to represent changes in uridine pool size. Restraint caused a reduction in DNA synthesis along the entire gastrointestinal tract and affected RNA metabolism in the stomach but not in the small intestine or colon. The changes in the stomach included early loss of total RNA followed by a decrease in the uptake of uridine5-3H. The latter appeared to coincide with the occurrence of gastric erosions. The importance of RNA metabolism in mucus production and in the maintenance of the mucosal cell population has been discussed in relation to the development of stress-induced gastric erosions. REFERENCES
BALM, M. E., SAMARTH,K.
D., HAMILTON, M. G., and PETERMANN, M. L. (1958). Role of the ribonucleoprotein particle in protein synthesis and the effects of growth hormone. J. Biol. Chem. 233, 1152-1155. BASERGA, R., ESTENSEN, R.D., and PETERSEN, R.O. (1967). Delayed inhibition of DNA synthesis in mouse jejunum by low doses of actinomycin D. J. Cell Physiol. 68, 177-184. BRODIE, D. A., and HANSON, H. M. (1960). A study of the factors involved in the production of gastric ulcers by the restraint technique. Gastroenterology 38, 353-360. BROWN, H. O., LEVINE, M. L., and LIPKIN, M. (1963). Inhibition of intestinal epithelial cell renewal and migration induced by starvation. Am. J. Physiol. 205, 868-872. BULLOUGH, W. S. (1952). Stress and epidermal mitotic activity. I. The effect of the adrenal hormones. J. Endocrinol. 8, 265-274. CLARK, C. M., NAISMATH, J. D., ~~~MuNRo, H. N. (1957). The influence of dietary protein on the incorporation of 14C-glycine and 32P into the ribonucleic acid of rat liver. Biochim. Biophys. Acta 23, 587-599. FELDMAN, S., BEHAR, A. J.. and BIRNBAUM, D. (1961). Gastric lesions following hypothalamic stimulation. Arch. Neural. 4, 308-317. GRAHAM, A. F., and RAKE, A. V. (1963). RNA Synthesis and turnover in mammalian cells propagated in vitro. Ann. Rev. Microbial. 17, 139-166. GRIFFITH, H. E. (1922). Trauma as a cause of chronic gastric ulcer. Lancet 203, 329-330. (1966). The effect of restraint on the content of HAKKINEN, I., HARTIALA, K., and LANG, H. acid polysaccharides of glandular gastric wall in rat. Acta Physiol. Scand. 66, 333-336. HOOPER, C. S., and BLAIR, M. (1958). The effect of starvation on epithelial renewal in the rat duodenum. Ezptl. Cell Res. 14, 175-181. KIM, Y. S., KERR, R., and LIPKIN, M. (1967). Cell proliferation during the development of stress erosions in mouse stomach. Nature 215, 1180-1181. KUMARESAN, P., and TURNER, C. W. (1966). Effect of various hormones on mammary gland growth of pregnant rats. Endocrinology 78, 396-399. LAHTIHARJU, A., RASANEN, T., and TEIR, H. (1964). Inhibition of DNA synthesis in various organs of the mouse following a single corticosteroid injection. Growth 28, 221-223. LAHTIHARJU, A., and RYTOMAA, T. (1967). DNA synthesis in fore and glandular stomach and in skin after nonspecific stress in mice. Exptl. Cell Res. 46, 593-596. LEBLOND, C. P., and CARRIERE, R. (1955). Effect of growth hormone and thyroxine on mitotic rate of intestinal mucosa of rat. Endocrinology 56, 261-266. MEJBAUM, W. (1939). Estimation of small amounts of pentose especially in derivatives of adenylic acid. 2. Physiol. Chem. 258, 117-120. MUNRO, H. N., and GOLDBERG, D. M. (1964). Effect of protein intake on protein and nucleic acid metabolism of the intestinal mucosal cell. 1% “The Role of the Gastrointestinal Tract in ProteinMetabolism,” (H. N. Mlmro, ed.), pp. 189-198, Davis, Philadelphia, Pennsylvania.
348
IMONDI,
BALIS,
AND
LIPKIN
A., and BERNHEIM,’ A. I. (1939). Acute postoperative esophageal, gastric, and ulcerations. Arch. Pathol. 28, 129-140. RASANEN, T. (1963). Fluctuations in the mitotic frequency of the glandular stomach and intestine of rat under the influence of ACTH, glucocorticords, stress, and heparin. Acta Physiol. Stand. 58, 201-210. RASANEN, T., CEDERBERG, A., and TASKINEN, E. (1966). The mitotic count in the gastointestinal epithelium and regenerating liver of heparinized rats. Gastroenterology 50,41-44. ROBERT, A., BAYER, R. B., and NEZAMIS, J. E. (1963). Gastric mucus content during development of stress ulcers. Gastroenterology 45, 740-751. ROSSI, G., BONFILS, S., LIEFFOGH, F., and LAMBLING, A. (1956). Technique nouvelle pour produire des ulcerations gastriques chez le rat blanc; I’ulceie de contrainte. Compf. Rend. Sot. Biol. 150, 2124-2126. SCHMIDT, G., and THANNHAUSER, S. J. (1945). A method for the determination of desoxyribonucleic acid, ribonucleic acid, and phosphoproteins in animal tissues. J. Biol. Chew 161, PENNER,
duodenal
83-89.
F. B. (1940). Removal of the impurities, nucleic acid and polysaccharide, from tuberculin protein. J. Biol. Chem. 133, 593-604. STEEL, R. G. D., and TORRIE, J. H. (1960). “Principles and Procedures of Statistics,” McGraw-Hill, New York. THIBODEAU, P. S., and THAYER, S. A. (1967). Effect of pregnancy and hormones on the activity of aspartate transcarbamylase and the level of nucleic acids in the mammary gland of the rat. Endocrinology 80, 505-509. WALKER, B. E., and LEBLOND, C. P. (1958). Cell proliferation and migration as revealed by radioautography after injection of thymidine-3H into male ram and mice. Am. J. Anat. SEIBERT,
106,
247-265.
AND
Nucleic
Acid
MOLECULAR
Metabolism
Mouse
during R.
ANTHONY
Department
of Medicine, For
(1968)
PATHOLOGY9,3%-348
IMONDI, Cornell
Cancer
in the Fasting M. University Research, Received
Gastrointestinal and
EARL
BALIS, Medical
New June
York,
Tract
of the
Restraint-Stress’ AND College New
MARTIN
LIPKIN
and Sloan-Kettering York
Institute
10021
21, 1968
The mucosal lining of the mammalian gastrointestinal tract undergoes profound changes during starvation and stress. Decreases in the number of cells lining the crypts and villi of rats starved for 5 days (Hooper and Blair, 1958) and in DNA synthesis in the intestinal mucosa of starved mice (Brown et al., 1963) have been reported. Munro and Goldberg (1964) reported that the feeding of protein-free diets to rats results in a decreased uptake of 3zP into DNA of intestinal mucosa but, had little effect on RNA metabolism. Subjecting animals or humans to stress such as restraint (Rossi et al., 1956)) operative procedures (Penner and Bernheim, 1939), or trauma (Griffith, 1922) produces acute erosions in the upper esophageal, gastric, and duodenal mucosa. Restraintstress has become a common method of producing gastric erosions experimentally. Recently, studies employing this technique have indicated that an inhibition of gastric epithelial cell proilferation precedes the development of erosions (Kim et al., 1967; Lahtiharju and Rytomaa, 1967). These studies have suggested that this inhibition of DNA synthesis and mitosis, with resulting failure to replace extruded epithelial cells, contributes to the development of the gastric erosions. The possible implication of altered nucleic acid metabolism in the development of the stress-induced gastric erosions has prompted an investigation of RNA and DNA metabolism in the gastrointestinal tract of mice subjected to stress. The present communication is a report of such studies and demonstrates differences in the uptake of nucleic acid precursors by cells in various regions of the gastrointestinal tract as a result of fasting and restraint METHODS Male CFW mice weighing 25-27 gm were used in these studies. Four experiments were conducted to measure the incorporation of thymidinemethy13H (TdR-3H; 16.5 c/m&!) into DNA after 16 hours (experiment 1) and 40 hours (experiment 2) and theincorporationof uridine-5-3H (URJH; 16.2 c/mM) into RNA after 16 hours (experiment 3) and 40 hours (experiment 4) of fasting and restraint. Both nucleosides were obtained from New England Nuclear, Boston. In each experiment four normal, four fasted, and four restrained animals were used. The normal mice were 1 This CA-08748;
work was supported by National Institutes I-F2-CA-37, 131.01; and K3-AM-4468. 339
of Health
Grants
and Awards
CA-08921
;
340
IMONDI,
BALIS,
AND
LIPBIN
kept in mouse cages with wood shavings until time of injection, and then transferred to wire-bottomed cages without food or water during the treatment period. Restrained mice were secured in wire mesh screen (Brodie and Hanson, 1960), also without food or water. At the end of each treatment period, each mouse was injected intraperitoneally with either 20 PC TdR-3H (experiments 1 and 2) or with 10 PC UR3H (experiments 3 and 4) and then returned to treatment. After one hour the mice were killed by cervical dislocation. The glandular stomach, duodenum (first 2.5 cm of small intestine), 2.5 cm of mid-jejunum, 2.5 cm of terminal ileum, and 2.5 cm of proximal colon were removed and homogenized with a Teflon pestle in 3 ml of icecold .Ol M Tris buffer (pH 7.5). The acid-insoluble material was precipitated by adding 1 ml of cold 2 N perchloric acid (PCA) and was then washed twice with 2 ml of cold .5 N PCA, once each with 2 ml of 75 %, 95 70, and 100 % ethanol, a 1: 1 mixture of ethanol-ether, and ether. UR-3H-containing material was hydrolyzed in 1 ml of .3 N NaOH at 37°C for 18 hours (Schmidt and Thannhauser, 1945). After precipitating DNA and protein with PCA, an aliquot of the RNA hydrolysate was assayed for RNA by a modification of the Mejbaum (1939) orcinol method. An appropriately diluted sample of RNA nucleotides was mixed with a concentrated HCl solution containing 0.5 % FeC& and 1% orcinol and was heated for 10 minutes at 100°C. The intensity of the green color was read at 670 rnp. Adenylic acid was used as a standard with appropriate correction. Another aliquot of the RNA hydrolysate was assayed for radioactivity in a Packard Tri-Carb Spectrometer with external standardization. The TdR-3H-containing samples were hydrolyzed in .Fi N PCA at 90°C for 15 minutes and assayed for DNA by the diphenylamine procedure of Seibert (1940) and for radioactivity as described above. Analysis of variance was performed and least significant differences between means were calculated (Steel and Torrie, 1960). The terms significant and highly significant are used throughout the text to denote differences between means at the .05 and .Ol probability levels, respectively. RESULTS DNA
Content of the tissues
The gastrointestinal mucosa of animals fasted for 16 hours and 40 hours revealed no gross abnormalities. In one of the animals restrained for 16 hours (experiment 3), there was blood in the lumen indicative of gastric erosions. All animals restrained for 40 hours had blood in the gastric lumen. DNA Content of the stomach did not decrease significantly after 40 hours of fasting or restraint (Figs. 1 and 2). The duodenum of the fasted and restrained animals did show a significant loss of DNA within 16 hours after treatment. The loss of DNA by the entire small intestine and colon was evident in the restrained mice after 40 hours. Incorporation
of TI~R-~H into DNA
The total amount of TdR-3H incorporated into DNA of the various tissues is presented in Figs. 1 and 2. Fasting for 16 hours had little effect on incorporation of
NUCLEIC
ACID
METABOLISM
DURING
pg DNA /tissue Normal
Fasted
Total TdR-3H /tissue
Restraint
Normal
341
STRESS
Fasted
(dpmx 10e3) Restraint
T 515 STOMACH
823-G DUODENUM
841
.
270 . i
JEJUNUM
462----F-
*
194 . +
ILEUM
740
275
COLON
. + 0
1. DNA Content and total amount tissue after 16 hours of fasting and restraint from normal (p < .05) ; l l , Different from .05). FIG.
0 of thymidine-3H incorporated into DNA of each (mean of 4 mice f standard error). l , Different normal (p < .Ol); +, Different from fasted (p <
TdR-3H, except for a significant decreasein duodenum. Fasting for 40 hours resulted in a decreased incorporation of TdR-3H in the ileum only. In the restrained mice, however, TdR-3H incorporation into DNA was markedly decreasedin all tissues at 16 and 40 hours. Except for the colon at 40 hours, the incorporation of TdR-3H was significantly lower in tissues of restrained mice than in those of both normal and fasted mice. In stomach, jejunum, and ileum the differences are highly significant. The specific activities of DNA (dpm/pg DNA) in the various tissues after 16 hours of treatment are presented in Table I. These data illustrate that the reduction in the incorporation of TdR-3H into DNA was greater than the loss of tissue DNA as a result of restraint. This was especially true in the stomach, where the specific activity was reduced 90% in 16 hours of restraint. Decreasesof 40-75% were observed in the other tissues.An unpublished observation in mice by one of us (A.R.I.) revealed that the incorporation of TdR-3H into DNA was reduced by 40% (p < .05) and 65 % (p < .Ol) in the stomach and jejunum, respectively, after 4 hours of restraint-stress.
342
IMONDI,
Incorporation
BALIS,
AND
LIPKIN
of UR-3H into RNA
Total RNA content was significantly lower in the stomach after 16 hours of restraint. Loss of RNA was not significant in the other tissues after 16 hours of treatment (Fig. 3). Incorporation of URJH was increased in all tissues of the fasted anipg DNA /tissue Normal
Fasted
Total TdR-3H /tissue
Restraint
Normal
Fasted
(dpm x 1O-3) Restraint
515 STOMACH
823
195
.
DUODENUM
. +
217
. JEJUNUM
.. +t 0
ILEUM
COLON
*'
FIG. 2. DNA Content and total amount of thymidine-3H incorporated into DNA of each tissue after 40 hours of fasting and restraint (mean of 4 mice f standard error). l , Different from normal (p < .05) ; 0 0, Different from normal (p < .Ol) ; +, Different from fasted (p < .05); + +, Different from fasted (p < .Ol). TABLE
I
INCORPORATION OF THYMIDINE-3H INTO DNA OF FASTING AND RESTRAINT (dpm/pg Normal
Organ
204 348 347 408 361
Stomach Duodenum Je j unum Ileum Colon D Mean
of 4 mice
f
f f f f f
standard
Fasted 4a 14 36 68 39
201 385 439 428 206 error.
f f f f f
AFTER
16 HOURS
DNA) Restraint
67 60 100 76 25
22 205 223 270 94
f f + + +
6 13 46 13 10
NUCLEIC
ACID
METABOLISM
pg RNA /tissue Normal
Fasted
Restraint
DURING
Total UR-3H/tissue Normal
343
STRESS
Fasted
(dpm x1F3) Restqint
1591 STOMACH 0
956 DUODENUM 0
+ JEJUNUM
. + 601 ILEUM 0
COLON
FIG. 3. RNA Content and total amount of uridine-5-3H tissue after 16 hours of fasting and restraint (mean of 4 mice from normal (p < .05); +, Different from fasted (p < .Ol).
incorporated f standard
into error).
RNA of each 0, Different
mals, and the differences were significant’ and highly significant in jejunum and ileum, respectively. When animals were restrained, however, there were no increases except in ileum. Incorporation of UR-3H into stomach RNA of the 16-hour restrained mice was lowest in the mouse with the gastric erosions. The specific activity of the RNA (Table II) was increased in all organs of the fasted group. A significant difference in the fasted jejunum reflects a very high uptake of UR-3H without a concomitant increase in total RNA. After 40 hours of treatment, the decrease in total stomach RNA in the restrained mice is highly significant compared to the normals and significant compared to the fasted mice (Fig. 4). After 40 hours of restraint, the incorporation of UR-3H was significantly decreased in the stomach but not in any other tissues. Fasting for 40 hours resulted in a slight but not significant increase in the uptake of UR-3H in duodenum and ileum. How-
344
IMONDI,
BALIS,
AND
TABLE
LIPKIN
II
INCORPORATION OF URIDINE-3H INTO RNA AFTER 16 HOURS OF FASTING AND RESTRAINT (dpm/pg RNA) Organ
Normal
Stomach Duodenum Jej unum Ileum Colon
16 16 28 22 19
f f f f f
Fasted 21 29 55 44 22
la 1 10 9 9
0 Mean of 4 mice f standard error. b Different from normal and restraint
f f f f f
Fasted
27 19 30 39 19
Total UR-3H/tissue
Restraint
1086
9 7 3 3 5
Normal
Fasted
(dpmx10-31 Restraint
.. + 0
f f f f f
37.1
1586 STOMACH
1 4 3b 8 1
(p < .05).
pg RNA /tissue Normal
Restraint
. 0
18.7
DUODENUM 0
0
1260 JEJUNUM 0
541 ILEUM 0 1185 COLON 0 FIG. 4. RNA Content and total amount tissue after 40 hours of fasting and restraint from normal (p < .05) ; 0 l , Different from .05).
of uridine-5-3H incorporated (mean of 4 mice f standard normal (p < .Ol) ; +, Different
into RNA of each error). l , Different from fasted (p <
ever, these increases, coupled with the change in total RNA in the tissues, result in specific activities (dpm/pg RNA) much higher in the duodenum and ileum of the fasted mice than those in either the normal or the restrained animals (Table III). The differences were significant and highly significant respectively.
NUCLEIC
ACID
METABOLISM
TABLE
DURING
345
STRESS
III
INCORPORATION OF URIDINFPH INTO RNA AFTER 40 HOURS OF FASTING AND RESTRAINT (dpm/pg RNA) Organ Stomach Duodenum Jej unum Ileum Colon
Normal 24 17 30 35 29
f f f f f
Fasted 4a 1 4 7 4
a Mean of 4 mice f standard error. b Different from normal and restraint c Different from normal and restraint
28 23 44 83 36
f f f f f
Restraint 4 2b 4 6” 6
25 13 32 36 32
f f f f f
5 3 9 7 4
(p < .05). (p < .Ol).
DISCUSSION Fasting and nucleic acid metabolism Fasting for up to 40 hours had no appreciable effect on either the amount or synthesis of DNA in the gastric wall. Only in duodenum did fasting result in decreased synthesis and loss of DNA. The reason for the early decrease in uptake of TdRJH by the fasted ileum is not clear since it was not evident at 40 hours. The RNA content of the gastrointestinal tissues was not affected by fasting. However, the uptake of UR-3H was increased in the small gut after 16 hours of fasting and returned to normal by 40 hours. Clark et al. (1957) reported a decrease in uptake of 32P by liver RNA in rats on the first day of protein-free feeding with an increase thereafter. These changes were attributed to a breakdown of liver RNA during protein depletion resulting in an enlargement of RNA precursor pools. Later, the pools shrink and the injected label is more extensively utilized. This apparent contrast in nucleic acid metabolism between intestine and liver is not unexpected in view of their marked differences in cellular activity. In addition, the rate of cell renewal is very rapid in the intestine but extremely slow in the liver. Stress and nucleic acid metabolism Previous studies using mitotic counts (Rasanen, 1963) and microradioautography (Kim et al., 1967; Lahtiharju and Rytomaa, 1967) have demonstrated that acute and chronic stress decrease the rate of cell division in stomach and epidermis of rodents. The present study confirms these findings and demonstrates that t#he decrease in DNA synthesis occurs throughout the gastrointestinal tract within 16 hours and even as early as 4 hours after the initiation of restraint-stress. Later, the total amount of DNA in duodenum and jejunum also decreased. Since the turnover rate of epithelial cells is somewhat greater in small intestine than in either st’omach or colon (Walker and J‘eblond, 195S), the marked reduction in DNA synthesis coupled with the continued cell loss results in earlier loss of DNA from t’he small intestine than from the other tissues. Metabolism of RNA in the small intestine and colon was less affected by restraint than was that of DNA. This was not true, however, for the stomach, which lost about 50 %. of its total RKA after 16 hours of restraint without decreased incorpora-
346
IMONDI,
BALE,
AND
LIPKIN
tion of UR-3H. Since ribosomal RNA (rRNA) constitutes about 80% of the total RNA and is not labeled to any extent by a l-hour pulse (Balis et al., 1958; Graham and Rake, 1963), it appears reasonable to suggest that most of the RNA lost by the restraint stomach was of ribosomal origin. The incorporation of UR-3H is probably indicative of a continuing synthesis of the rapidly metabolized messenger RNA (mRNA) . The specific mechanisms initiating this alteration in nucleic acid metabolism are not known. Various hormones have been shown to influence nucleic acid synthesis in mammary tissue (Kumaresan and Turner, 1966; Thibodeau and Thayer, 1967), while growth hormone has been shown by Leblond and Carriere (1955) to overcome the decrease in cell proliferation in the intestinal mucosa resulting from hypophysectomy. It has been suggested that adrenal hormones are responsible for decreasing mitotic activity in the epidermis of stressed animals (Bullough, 1952), while corticosteroids cause a greater reduction in mitotic activity in the stomach than in the duodenum (Lahtiharju et al., 1964). Heparin also depresses mitotic activity in the stomach but has no effect on cell proliferation in the small bowel or colon (Rasanen et al., 1966). Experimental lesions in the hypothalamus also cause erosions and hemorrhage in cats (Feldman et al., 1961) and, recent unpublished observations in this laboratory indicate that these result in a depression of cell proliferation in the stomach of rats. Nucleic acid metabolzsm and the development of gastric erosions The marked depression in DNA synthesis, accompanied by a loss of RNA, was followed by the development of gastric erosions. The eventual decrease in synthesis of RNA detected in gastric tissue after 40 hours of stress accompanied the occurrence of erosions in the gastric mucosa. A single animal which manifested erosions after 16 hours of restraint also incorporated less of UR-3H into RNA. These findings suggest that the initial loss of rRNA and the ensuing decrease in the synthesis of mRNA are part of a sequence of events leading to the destruction of epithelial cells of the gastric mucosa. Ribosomes are the site of protein synthesis and it follows that their loss of RNA should result in impaired protein synthesis. Although a relationship between ribosomal function and muco-protein synthesis has not been demonstrated, it is likely that some connection does exist either directly or through the enzymes involved in mucus production. Therefore, loss of rRNA could lead to a decrease in the synthesis of mucus. Indeed, a decrease in gastric mucus has been associated with the appearance of gastric erosions in rats (Hakkinen et al., 1966; Robert et al., 1963) and in mice (Kim et al., 1967). The relationship between RNA and mucus production on one hand and the appearance of erosions on the other is particularly significant in view of the results observed in the small intestine and colon. Here the synthesis of DNA was reduced, but no marked decreases in RNA content or synthesis were noted and erosions were not observed in these regions. It is also known that RNA and protein synthesis are prerequisites for synthesis of DNA (Baserga et al., 1967), which, in turn, is needed for mitosis. Hence, RNA synthesis is important for the maintenance of normal cell replacement, and any’im-
NUCLEIC
ACID
METABOLISM
DURING
347
STRESS
pairment in this process will lead to decreased cell proliferation the tissue to compensate for the loss of cells.
and the inability
of
SUMMARY The uptake of thymidine-methyl-3H and uridine-5-3H into DNA and RNA of gastrointestinal tissues of mice has been measured after 16 and 40 hours of fasting and restraint. Fasting had little effect on DNA or RNA metabolism in the gastrointestinal tract except for increases in the uptake of uridine in the small intestine. These increases are thought to represent changes in uridine pool size. Restraint caused a reduction in DNA synthesis along the entire gastrointestinal tract and affected RNA metabolism in the stomach but not in the small intestine or colon. The changes in the stomach included early loss of total RNA followed by a decrease in the uptake of uridine5-3H. The latter appeared to coincide with the occurrence of gastric erosions. The importance of RNA metabolism in mucus production and in the maintenance of the mucosal cell population has been discussed in relation to the development of stress-induced gastric erosions. REFERENCES
BALM, M. E., SAMARTH,K.
D., HAMILTON, M. G., and PETERMANN, M. L. (1958). Role of the ribonucleoprotein particle in protein synthesis and the effects of growth hormone. J. Biol. Chem. 233, 1152-1155. BASERGA, R., ESTENSEN, R.D., and PETERSEN, R.O. (1967). Delayed inhibition of DNA synthesis in mouse jejunum by low doses of actinomycin D. J. Cell Physiol. 68, 177-184. BRODIE, D. A., and HANSON, H. M. (1960). A study of the factors involved in the production of gastric ulcers by the restraint technique. Gastroenterology 38, 353-360. BROWN, H. O., LEVINE, M. L., and LIPKIN, M. (1963). Inhibition of intestinal epithelial cell renewal and migration induced by starvation. Am. J. Physiol. 205, 868-872. BULLOUGH, W. S. (1952). Stress and epidermal mitotic activity. I. The effect of the adrenal hormones. J. Endocrinol. 8, 265-274. CLARK, C. M., NAISMATH, J. D., ~~~MuNRo, H. N. (1957). The influence of dietary protein on the incorporation of 14C-glycine and 32P into the ribonucleic acid of rat liver. Biochim. Biophys. Acta 23, 587-599. FELDMAN, S., BEHAR, A. J.. and BIRNBAUM, D. (1961). Gastric lesions following hypothalamic stimulation. Arch. Neural. 4, 308-317. GRAHAM, A. F., and RAKE, A. V. (1963). RNA Synthesis and turnover in mammalian cells propagated in vitro. Ann. Rev. Microbial. 17, 139-166. GRIFFITH, H. E. (1922). Trauma as a cause of chronic gastric ulcer. Lancet 203, 329-330. (1966). The effect of restraint on the content of HAKKINEN, I., HARTIALA, K., and LANG, H. acid polysaccharides of glandular gastric wall in rat. Acta Physiol. Scand. 66, 333-336. HOOPER, C. S., and BLAIR, M. (1958). The effect of starvation on epithelial renewal in the rat duodenum. Ezptl. Cell Res. 14, 175-181. KIM, Y. S., KERR, R., and LIPKIN, M. (1967). Cell proliferation during the development of stress erosions in mouse stomach. Nature 215, 1180-1181. KUMARESAN, P., and TURNER, C. W. (1966). Effect of various hormones on mammary gland growth of pregnant rats. Endocrinology 78, 396-399. LAHTIHARJU, A., RASANEN, T., and TEIR, H. (1964). Inhibition of DNA synthesis in various organs of the mouse following a single corticosteroid injection. Growth 28, 221-223. LAHTIHARJU, A., and RYTOMAA, T. (1967). DNA synthesis in fore and glandular stomach and in skin after nonspecific stress in mice. Exptl. Cell Res. 46, 593-596. LEBLOND, C. P., and CARRIERE, R. (1955). Effect of growth hormone and thyroxine on mitotic rate of intestinal mucosa of rat. Endocrinology 56, 261-266. MEJBAUM, W. (1939). Estimation of small amounts of pentose especially in derivatives of adenylic acid. 2. Physiol. Chem. 258, 117-120. MUNRO, H. N., and GOLDBERG, D. M. (1964). Effect of protein intake on protein and nucleic acid metabolism of the intestinal mucosal cell. 1% “The Role of the Gastrointestinal Tract in ProteinMetabolism,” (H. N. Mlmro, ed.), pp. 189-198, Davis, Philadelphia, Pennsylvania.
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